Processing biomass

ABSTRACT

Biomass (e.g., plant biomass, animal biomass, and municipal waste biomass) is processed to produce useful products, such as fuels. For example, systems can use feedstock materials, such as cellulosic and/or lignocellulosic materials, to proceed ethanol and/or butanol, e.g., by fermentation.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. Ser. No. 14/169,174, filedJan. 31, 2014, which is a continuation of U.S. Ser. No. 13/935,065 filedJul. 3, 2013, which is now U.S. Pat. No. 8,709,771, issued Apr. 29,2014, which is a continuation of U.S. Ser. No. 13/554,392 filed Jul. 20,2012, which is now U.S. Pat. No. 8,518,683, issued Aug. 27, 2013, whichis a continuation of U.S. Ser. No. 12/417,840, filed Apr. 3, 2009, whichis now U.S. Pat. No. 8,236,535 issued Aug. 7, 2012, which claimedpriority from U.S. Provisional Application Ser. No. 61/049,407, filedApr. 30, 2008. The entirety of each of these applications isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to processing biomass, and products madetherefrom.

BACKGROUND

Various carbohydrates, such as cellulosic and lignocellulosic materials,e.g., in fibrous form, are produced, processed, and used in largequantities in a number of applications. Often such materials are usedonce, and then discarded as waste, or are simply considered to be wastematerials, e.g., sewage, bagasse, sawdust, and stover.

Various cellulosic and lignocellulosic materials, their uses, andapplications have been described in U.S. Pat. Nos. 7,307,108, 7,074,918,6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105; and in variouspatent applications, including “FIBROUS MATERIALS AND COMPOSITES,”PCT/US2006/010648, filed on Mar. 23, 2006, and “FIBROUS MATERIALS ANDCOMPOSITES,” U.S. Patent Application Publication No. 2007/0045456.

SUMMARY OF THE INVENTION

Generally, the inventions described herein relate tocarbohydrate-containing materials (e.g., biomass materials orbiomass-derived materials, such as starchy materials, cellulosicmaterials, lignocellulosic materials, or biomass materials that are orthat include significant amounts of low molecular weight sugars (e.g.,monosaccharides, disaccharides or trisaccharides), methods of making andprocessing such materials to change their structure, e.g., functionalizethese materials with one or more desired types and amounts of functionalgroups, and products made from the structurally changed materials. Forexample, many of the methods described herein can provide cellulosicand/or lignocellulosic materials that have a lower molecular weightand/or crystallinity relative to a native material. Many of the methodsprovide materials that can be more readily utilized by a variety ofmicroorganisms to produce useful products, such as hydrogen, alcohols(e.g., ethanol or butanol), organic acids (e.g., acetic acid),hydrocarbons, co-products (e.g., proteins) or mixtures of any of these.

In some instances, functionalized biomass is more soluble and morereadily utilized by microorganisms in comparison to biomass that has notbeen functionalized. In addition, many of the functionalized materialsdescribed herein are less prone to oxidation and can have enhancedlong-term stability (e.g., oxidation in air under ambient conditions).Many of the products obtained, such as ethanol or n-butanol, can beutilized as a fuel for powering cars, trucks, tractors, ships or trains,e.g., as an internal combustion fuel or as a fuel cell feedstock. Manyof the products obtained can also be utilized to power aircraft, such asplanes, e.g., having jet engines or helicopters. In addition, theproducts described herein can be utilized for electrical powergeneration, e.g., in a conventional steam generating plant or in a fuelcell plant.

In one aspect, the invention features a method that includes convertinga low molecular weight sugar, or a material that includes a lowmolecular weight sugar, in a mixture with a biomass, a microorganism,and a solvent or a solvent system, e.g., water or a mixture of water andan organic solvent, to a product. Examples of solvents or solventsystems include water, hexane, hexadecane, glycerol, chloroform,toluene, ethyl acetate, petroleum ether, liquefied petroleum gas (LPG),ionic liquids and mixtures thereof. The solvent or solvent system can bein the form of a single phase or two or more phases. The biomass can be,e.g., in fibrous form.

In some instances, having a biomass material (e.g., treated by anymethod described herein or untreated) present during production of aproduct, such as ethanol, can enhance the production rate of theproduct. Without wishing to be bound by any particular theory, it isbelieved that having a solid present, such as a high surface area and/orhigh porosity solid, can increase reaction rates by increasing theeffective concentration of solutes and providing a substrate on whichreactions can occur. For example, an irradiated or an un-irradiatedbiomass material, e.g., a paper fiber, can be added to a fermentationprocess, such as during a corn-ethanol fermentation or a sugarcaneextract fermentation, to increase the rate of production by 10, 15, 20,30, 40, 50, 75, 100 percent or more, e.g., 150 percent. The biomassmaterial can have a high surface area, high porosity, and/or low bulkdensity. In some embodiments, the biomass is present in the mixture fromabout 0.5 percent to about 50 percent by weight, such as between about 1percent and about 25 percent by weight, or between about 2 percent andabout 12.5 percent by weight. In other embodiments, the biomass ispresent in amounts greater than about 0.5 percent by weight, such asgreater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or even greater than about10 percent by weight. For example, in some embodiments, an oxidized,sonicated, steam exploded and/or pyrolyzed biomass material, such as apaper or cotton fiber, can be added to a low molecular weight sugarfermentation process, e.g., to enhance fermentation rate and output.

Some implementations include one or more of the following features. Thebiomass may comprise a fibrous material. Converting can include allowingthe microorganism to convert at least a portion of the low molecularweight sugar to ethanol. For example, converting can comprisefermentation. The microorganism can comprise a yeast, e.g., selectedfrom the group consisting of S. cerevisiae and P. stipitis, or abacterium such as Zymomonas mobilis. Converting can exhibit a %performance of at least 140%, in some cases at least 170%.

The method can further include irradiating the fibrous biomass prior tomixing, e.g., with ionizing radiation, for example at a total dosage ofat least 5 Mrad. Irradiating can be performed using a particle beam.Irradiating can be conducted under conditions selected to reduce themolecular weight of the biomass.

The biomass can have a bulk density of less than about 0.5 g/cm³. Thebiomass can have a BET surface area of greater than 0.25 m²/g, and/or alength to diameter ratio of at least 5. The biomass can have a porositygreater than 50%.

The method can further include physically preparing the biomass, e.g.,by shearing, or by reducing the size of the biomass by stone grinding,mechanical ripping or tearing, pin grinding, or air attrition milling.The biomass can have internal fibers, and can have been sheared to anextent that its internal fibers are substantially exposed.

The biomass may be or may include a cellulosic or lignocellulosicmaterial. For example, the biomass can be selected from the groupconsisting of paper, paper products, paper waste, wood, particle board,sawdust, agricultural waste, sewage, silage, grasses, rice hulls,bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corncobs, corn stover, switchgrass, alfalfa, hay, rice hulls, coconut hair,cotton, seaweed, algae, and mixtures thereof.

The method may further include subjecting the biomass to enzymatichydrolysis, and in some cases converting the hydrolyzed material to theproduct.

In another aspect, the invention features a method for dissolving acellulosic or lignocellulosic material, the method comprising combininga cellulosic or lignocellulosic material with a solvent systemcomprising DMSO and a salt.

Solvent systems for cellulosic and lignocellulosic materials includeDMSO-salt systems. Such systems include, for example, DMSO incombination with a lithium, magnesium, potassium, sodium or zinc salt.Lithium salts include LiCl, LiBr, Li, lithium perchlorate and lithiumnitrate. Magnesium salts include magnesium nitrate and magnesiumchloride. Potassium salts include potassium iodide and nitrate. Examplesof sodium salts include sodium iodide and nitrate. Examples of zincsalts include zinc chloride and nitrate. Any salt can be anhydrous orhydrated. Typical loadings of the salt in the DMSO are between about 1and about 50 percent, e.g., between about 2 and 25, between about 3 and15 or between about 4 and 12.5 percent by weight.

In other implementations, the salt can be a fluoride salt, e.g.tetrabutyl ammonium fluoride. The method can further include irradiatingthe cellulosic or lignocellulosic material. The cellulosic orlignocellulosic material can be selected from the group consisting ofpaper, paper products, paper waste, wood, particle board, sawdust,agricultural waste, sewage, silage, grasses, rice hulls, bagasse,cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, cornstover, switchgrass, alfalfa, hay, rice hulls, coconut hair, cotton,seaweed, algae, and mixtures thereof. In some cases, the cellulosic orlignocellulosic material has a bulk density of less than about 0.5 g/cm³(prior to addition to the solvent system) and a porosity of at least50%.

Materials are disclosed herein that include a plurality of saccharideunits arranged in a molecular chain, wherein from about 1 out of every 2to about 1 out of every 250 saccharide units includes a carboxylic acidgroup, or an ester or salt thereof. In another aspect, materials includea plurality of such molecular chains. For example, about 1 out of every8, 1 out of every 10, 1 out of every 50, or 1 out of every 100saccharide units of each chain can include a carboxylic acid group, oran ester or salt thereof. In some embodiments, the saccharide units caninclude 5 or 6 carbon saccharide units. Each chain can have betweenabout 10 and about 200 saccharide units, e.g., between about 10 andabout 100 or between about 10 and about 50. For example, each chain caninclude hemicellulose or cellulose. In some embodiments, each chain alsoincludes saccharide units that include nitroso, nitro, or nitrilegroups.

In some embodiments, the average molecular weight of the materialsrelative to PEG standards can be from about 1,000 to about 1,000,000,such as between 1,500 and 200,000 or 2,000 and 10,000. For example, theaverage molecular weight of the materials relative to PEG standards canbe less than about 10,000.

Methods of changing a molecular and/or a supramolecular structure of abiomass feedstock are disclosed herein that include 1) irradiating thebiomass feedstock with radiation, such as photons, electrons or ions ofsufficient energy to ionize the biomass feedstock, to provide a firstlevel of radicals, e.g., which are detectable with an electron spinresonance spectrometer; 2) quenching the radicals to an extent that theradicals are at a second level lower than the first level, such as at alevel that is no longer detectable with the electron spin resonancespectrometer, e.g., such as at a level of less than about 10¹⁴ spins;and 3) processing the irradiated biomass feedstock to produce a product.If desired, prior to irradiation and/or after irradiation, the biomassfeedstock can be prepared by reducing one or more dimensions ofindividual pieces of the biomass feedstock.

In some implementations, the processing step includes making a product,such as a fuel, such as a combustible fuel, such as a motor, an aviationfuel or a fuel cell fuel, e.g., for generating electricity, byconverting the irradiated biomass feedstock with a microorganism havingthe ability to convert at least a portion, e.g., at least about 1percent by weight, of the biomass to the product.

In some embodiments, irradiating is performed on the biomass feedstockwhile the biomass feedstock is exposed to air, nitrogen, oxygen, helium,or argon. In some embodiments, pretreatment can include pretreating thebiomass feedstock with steam explosion.

In some embodiments, the method further includes reducing one or moredimensions of individual pieces of biomass, for example, by shearing,wet or dry grinding, cutting, squeezing, compressing or mixtures of anyof these processes. For example, shearing can be performed with a rotaryknife cutter. The shearing can produce fibers having an averagelength-to-diameter ratio of greater than 5/1. In some embodiments, theprepared biomass can have a BET surface area of greater than 0.25 m²/g.The biomass can be sheared to an extent that internal fibers of thebiomass are substantially exposed. The biomass can be sheared to anextent that it has a bulk density of less than about 0.35 g/cm³

In some embodiments, the process does not include hydrolyzing thebiomass with an acid or a base. For example, at least about seventypercent by weight of the biomass can be un-hydrolyzed, e.g., at least at95 percent by weight of the biomass has not been hydrolyzed. In specificembodiments, substantially none of the biomass has been hydrolyzed.

In some embodiments, irradiation is performed on biomass in which lessthan about 25 percent by weight of the biomass is wetted with a liquid,such as water. Specifically, in some embodiments, at least onepretreatment method is performed on biomass in which substantially noneof the biomass is wetted with a liquid, such as water. The biomass canhave, e.g., less than about five percent by weight retained water,measured at 25° C. and at fifty percent relative humidity.

In some embodiments, irradiation is performed on biomass in which lessthan about 25 percent by weight of the biomass is in a swollen state,the swollen state being characterized as having a volume of more thanabout 2.5 percent higher than an unswollen state. In other embodiments,the biomass is mixed with or includes a swelling agent.

Pressure can be utilized in any of the methods described herein. Forexample, irradiation can be performed on the biomass under a pressure ofgreater than about 2.5 atmospheres, such as greater than 5 or 10atmospheres.

Examples of biomass feedstock include paper, paper products, paperwaste, wood, particle board, sawdust, agricultural waste, sewage,silage, grasses, rice hulls, bagasse, cotton, jute, hemp, flax, bamboo,sisal, abaca, straw, corn cobs, corn stover, switchgrass, alfalfa, hay,rice hulls, coconut hair, cotton, synthetic celluloses, seaweed, algae,or mixtures of these. The biomass can be or can include a natural or asynthetic material.

Examples of fuels include one or more of hydrogen, alcohols, andhydrocarbons. For example, the alcohols can be ethanol, n-propanol,isopropanol, n-butanol, or mixtures of these.

Irradiation can be, e.g., performed utilizing an ionizing radiation,such as gamma rays, a beam of electrons, or ultraviolet C radiationhaving a wavelength of from about 100 nm to about 280 nm. Irradiationcan be performed using multiple applications of radiation. The ionizingradiation can include electron beam radiation. For example, theradiation can be applied at a total dose of between about 10 Mrad andabout 150 Mrad, such as at a dose rate of about 0.5 to about 10Mrad/day, or 1 Mrad/s to about 10 Mrad/s. In some embodiments,irradiating includes applying two or more radiation sources, such asgamma rays and a beam of electrons.

In some embodiments, the biomass includes a first cellulose having afirst number average molecular weight and the carbohydrate materialcomprises a second cellulose having a second number average molecularweight lower than the first number average molecular weight. Forexample, the second number average molecular weight is lower than thefirst number average molecular weight by more than about one-fold. Insome embodiments, the first cellulose has a first crystallinity, and thesecond cellulose has a second crystallinity lower than the firstcrystallinity. For example, the second crystallinity can be lower thanthe first crystallinity by more than about 10 percent.

In some embodiments, the first cellulose can have a first level ofoxidation and the second cellulose has a second level of oxidationhigher than the first level of oxidation.

The biomass material can further include a buffer, such as sodiumbicarbonate or ammonium chloride, an electrolyte, such as potassiumchloride or sodium chloride a growth factor, such as biotin and/or abase pair such as uracil, a surfactant, a mineral, or a chelating agent.

In some embodiments, the methods include pretreating with one or moreother pretreatment methods in addition to irradiation. For example, thetwo or more different pretreatment methods can include radiation andsonication, radiation and oxidation, and radiation and pyrolization.Optionally, pretreating the biomass can include steam explosion

To further aid in the reduction of the molecular weight of the biomass,an enzyme, e.g., a cellulolytic enzyme, or a chemical, e.g., sodiumhypochlorite, an acid, a base or a swelling agent, can be utilized withany method described herein. The enzyme and/or chemical treatment canoccur before, during or after irradiation or other pretreatment.

When a microorganism is utilized, it can be a natural microorganism oran engineered microorganism. For example, the microorganism can be abacterium, e.g., a cellulolytic bacterium, a fungus, e.g., a yeast, aplant or a protist, e.g., an algae, a protozoa or a fungus-like protist,e.g., a slime mold. When the organisms are compatible, mixtures may beutilized. Generally, various microorganisms can produce a number ofuseful products, such as a fuel, by operating on, e.g., fermenting thematerials. For example, alcohols, organic acids, hydrocarbons, hydrogen,proteins or mixtures of any of these materials can be produced byfermentation or other processes.

Examples of products that may be produced using the methods disclosedherein include mono- and polyfunctional C1-C6 alkyl alcohols, mono- andpoly-functional carboxylic acids, C1-C6 hydrocarbons, and combinationsthereof. Specific examples of suitable alcohols include methanol,ethanol, propanol, isopropanol, butanol, ethylene glycol, propyleneglycol, 1,4-butane diol, glycerin, and combinations thereof. Specificexample of suitable carboxylic acids include formic acid, acetic acid,propionic acid, butyric acid, valeric acid, caproic acid, palmitic acid,stearic acid, oxalic acid, malonic acid, succinic acid, glutaric acid,oleic acid, linoleic acid, glycolic acid, lactic acid,gamma-hydroxybutyric acid, and combinations thereof. Examples ofsuitable hydrocarbons include methane, ethane, propane, pentane,n-hexane, and combinations thereof. Many of these products may be usedas fuels. Other products are described in U.S. Provisional ApplicationSer. No. 61/139,453, the full disclosure of which is incorporated byreference herein. Products or co-products produced can be productsintended to be used as produced or the products produced can beintermediates for any other process described herein or any processdescribed in any application incorporated by reference herein.

Examples of microorganisms that may be used to produce useful productsinclude bacteria, yeasts, or combinations thereof. For example, themicroorganism can be a bacterium, e.g., a cellulolytic bacterium, afungus, e.g., a yeast, a plant or a protist, e.g., an algae, a protozoaor a fungus-like protist, e.g., a slime mold.

In any of the methods disclosed herein, radiation may be applied from adevice that is in a vault.

The term “fibrous material,” as used herein, is a material that includesnumerous loose, discrete and separable fibers. For example, a fibrousmaterial can be prepared from a bleached Kraft paper fiber source byshearing, e.g., with a rotary knife cutter.

The term “screen,” as used herein, means a member capable of sievingmaterial according to size. Examples of screens include a perforatedplate, cylinder or the like, or a wire mesh or cloth fabric.

The term “pyrolysis,” as used herein, means to break bonds in a materialby the application of heat energy. Pyrolysis can occur while the subjectmaterial is under vacuum, or immersed in a gaseous material, such as anoxidizing gas, e.g., air or oxygen, or a reducing gas, such as hydrogen.

Oxygen content is measured by elemental analysis by pyrolyzing a samplein a furnace operating at 1300° C. or above.

The term “biomass” includes any non-fossilized, i.e., renewable, organicmatter. The various types of biomass include plant biomass (definedbelow), microbial biomass, animal biomass (any animal by-product, animalwaste, etc.) and municipal waste biomass (residential and lightcommercial refuse with recyclables such as metal and glass removed). Theterm biomass also includes virgin or post-consumer cellulosic materials,such as rags and towels fabricated from cotton or a cotton blend.

The terms “plant biomass” and “lignocellulosic biomass” refer tovirtually any plant-derived organic matter (woody or non-woody). Plantbiomass can include, but is not limited to, agricultural or food crops(e.g., sugarcane, sugar beets or corn kernels) or an extract therefrom(e.g., sugar from sugarcane and corn starch from corn), agriculturalcrops and agricultural crop wastes and residues such as corn stover,wheat straw, rice straw, sugar cane bagasse, cotton and the like. Plantbiomass further includes, but is not limited to, trees, woody energycrops, wood wastes and residues such as softwood forest thinnings, barkywastes, sawdust, paper and pulp industry waste streams, wood fiber, andthe like. Additionally, grass crops, such as switchgrass and the likehave potential to be produced on a large-scale as another plant biomasssource. For urban areas, the best potential plant biomass feedstockincludes yard waste (e.g., grass clippings, leaves, tree clippings, andbrush) and vegetable processing waste.

“Lignocellulosic feedstock,” is any type of plant biomass such as, butnot limited to, non-woody plant biomass, cultivated crops, such as, butnot limited to, grasses, for example, but not limited to, C4 grasses,such as switchgrass, cord grass, rye grass, miscanthus, reed canarygrass, or a combination thereof, or sugar processing residues such asbagasse, or beet pulp, agricultural residues, for example, soybeanstover, corn stover, rice straw, rice hulls, barley straw, corn cobs,wheat straw, canola straw, rice straw, oat straw, oat hulls, corn fiber,recycled wood pulp fiber, sawdust, hardwood, for example, aspen wood andsawdust, softwood, or a combination thereof. Further, thelignocellulosic feedstock may include cellulosic waste material such as,but not limited to, newsprint, cardboard, sawdust, and the like.

Lignocellulosic feedstock may include one species of fiber oralternatively, lignocellulosic feedstock may include a mixture of fibersthat originate from different lignocellulosic feedstocks. Furthermore,the lignocellulosic feedstock may comprise fresh lignocellulosicfeedstock, partially dried lignocellulosic feedstock, fully driedlignocellulosic feedstock or a combination thereof.

For the purposes of this disclosure, carbohydrates are materials thatare composed entirely of one or more saccharide units or that includeone or more saccharide units. The saccharide units can be functionalizedabout the ring with one or more functional groups, such as carboxylicacid groups, amino groups, nitro groups, nitroso groups or nitrilegroups and still be considered carbohydrates. Carbohydrates can bepolymeric (e.g., equal to or greater than 10-mer, 100-mer, 1,000-mer,10,000-mer, or 100,000-mer), oligomeric (e.g., equal to or greater thana 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer or 10-mer), trimeric,dimeric, or monomeric. When the carbohydrates are formed of more than asingle repeat unit, each repeat unit can be the same or different.

Examples of polymeric carbohydrates include cellulose, xylan, pectin,and starch, while cellobiose and lactose are examples of dimericcarbohydrates. Examples of monomeric carbohydrates include glucose andxylose

Carbohydrates can be part of a supramolecular structure, e.g.,covalently bonded into the structure. Examples of such materials includelignocellulosic materials, such as those found in wood.

A starchy material is one that is or includes significant amounts ofstarch or a starch derivative, such as greater than about 5 percent byweight starch or starch derivative. For purposes of this disclosure, astarch is a material that is or includes an amylose, an amylopectin, ora physical and/or chemical mixture thereof, e.g., a 20:80 or 30:70percent by weight mixture of amylose to amylopectin. For example, rice,corn, and mixtures thereof are starchy materials. Starch derivativesinclude, e.g., maltodextrin, acid-modified starch, base-modified starch,bleached starch, oxidized starch, acetylated starch, acetylated andoxidized starch, phosphate-modified starch, genetically-modified starchand starch that is resistant to digestion.

For purposes of this disclosure, a low molecular weight sugar is acarbohydrate or a derivative thereof that has a formula weight(excluding moisture) that is less than about 2,000, e.g., less thanabout 1,800, 1,600, less than about 1,000, less than about 500, lessthan about 350 or less than about 250. For example, the low molecularweight sugar can be a monosaccharide, e.g., glucose or xylose, adisaccharide, e.g., cellobiose or sucrose, or a trisaccharide.

A combustible fuel is a material capable of burning in the presence ofoxygen. Examples of combustible fuels include ethanol, n-propanol,n-butanol, hydrogen and mixtures of any two or more of these.

Swelling agents as used herein are materials that cause a discernableswelling, e.g., a 2.5 percent increase in volume over an unswollen stateof cellulosic and/or lignocellulosic materials, when applied to suchmaterials as a solution, e.g., a water solution. Examples includealkaline substances, such as sodium hydroxide, potassium hydroxide,lithium hydroxide and ammonium hydroxides, acidifying agents, such asmineral acids (e.g., sulfuric acid, hydrochloric acid and phosphoricacid), salts, such as zinc chloride, calcium carbonate, sodiumcarbonate, benzyltrimethylammonium sulfate, and basic organic amines,such as ethylene diamine.

A “sheared material,” as used herein, is a material that includesdiscrete fibers in which at least about 50% of the discrete fibers havea length/diameter (LID) ratio of at least about 5, and that has anuncompressed bulk density of less than about 0.6 g/cm³. A shearedmaterial is thus different from a material that has been cut, chopped orground.

Changing a molecular structure of a biomass feedstock, as used herein,means to change the chemical bonding arrangement, such as the type andquantity of functional groups or conformation of the structure. Forexample, the change in the molecular structure can include changing thesupramolecular structure of the material, oxidation of the material,changing an average molecular weight, changing an average crystallinity,changing a surface area, changing a degree of polymerization, changing aporosity, changing a degree of branching, grafting on other materials,changing a crystalline domain size, or an changing an overall domainsize.

This application incorporates by reference herein the entire contents ofInternational Application No. PCT/US2007/022719, filed on Oct. 26, 2007.The full disclosures of each of the following U.S. patent applications,are incorporated by reference herein: U.S. application Ser. No.12/417,707, filing date Apr. 3, 2009, (now U.S. Pat. No. 7,867,358),U.S. application Ser. No. 12/417,720, filing date Apr. 3, 2009, (nowU.S. Pat. No. 7,846,295), U.S. application Ser. No. 12/417,699, filingdate Apr. 3, 2009, (now U.S. Pat. No. 7,931,784), U.S. application Ser.No. 12/417,731, filing date Apr. 3, 2009, published as US 2010/009324,U.S. application Ser. No. 12/417,900, filing date Apr. 3, 2009,published as US 2010/0124583, U.S. application Ser. No. 12/417,880,filing date Apr. 3, 2009, (now U.S. Pat. No. 8,212,087), U.S.application Ser. No. 12/417,723, filing date Apr. 3, 2009, published asUS 2009/0312537, U.S. application Ser. No. 12/417,786, filing date Apr.3, 2009, (now U.S. Pat. No. 8,025,098), and U.S. application Ser. No.12/417,904, filing date Apr. 3, 2009, (now U.S. Pat. No. 7,867,359).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram illustrating conversion of biomass intoproducts and co-products.

FIG. 2 is block diagram illustrating conversion of a fiber source into afirst and second fibrous material.

FIG. 3 is a cross-sectional view of a rotary knife cutter.

FIG. 4 is block diagram illustrating conversion of a fiber source into afirst, second and third fibrous material.

FIG. 5 is block diagram illustrating densification of a material.

FIG. 6 is a perspective view of a pellet mill.

FIG. 7A is a densified fibrous material in pellet form.

FIG. 7B is a transverse cross-section of a hollow pellet in which acenter of the hollow is in-line with a center of the pellet.

FIG. 7C is a transverse cross-section of a hollow pellet in which acenter of the hollow is out of line with the center of the pellet.

FIG. 7D is a transverse cross-section of a tri-lobal pellet.

FIG. 8 is a block diagram illustrating a treatment sequence forprocessing feedstock.

FIG. 9 is a perspective, cut-away view of a gamma irradiator housed in aconcrete vault.

FIG. 10 is an enlarged perspective view of region R of FIG. 9.

FIG. 11A is a block diagram illustrating an electron beam irradiationfeedstock pretreatment sequence.

FIG. 11B is a schematic representation of biomass being ionized, andthen oxidized or quenched.

FIG. 12 is a schematic view of a system for sonicating a process streamof cellulosic material in a liquid medium.

FIG. 13 is a schematic view of a sonicator having two transducerscoupled to a single horn.

FIG. 14 is a block diagram illustrating a pyrolytic feedstockpretreatment system.

FIG. 15 is a cross-sectional side view of a pyrolysis chamber.

FIG. 16 is a cross-sectional side view of a pyrolysis chamber.

FIG. 17 is a cross-sectional side view of a pyrolyzer that includes aheated filament.

FIG. 18 is a schematic cross-sectional side view of a Curie-Pointpyrolyzer.

FIG. 19 is a schematic cross-sectional side view of a furnace pyrolyzer.

FIG. 20 is a schematic cross-sectional top view of a laser pyrolysisapparatus.

FIG. 21 is a schematic cross-sectional top view of a tungsten filamentflash pyrolyzer.

FIG. 22 is a block diagram illustrating an oxidative feedstockpretreatment system.

FIG. 23 is block diagram illustrating a general overview of the processof converting a fiber source into a product, e.g., ethanol.

FIG. 24 is a cross-sectional view of a steam explosion apparatus.

FIG. 25 is a schematic cross-sectional side view of a hybrid electronbeam/sonication device.

FIG. 26 is a scanning electron micrograph of a fibrous material producedfrom polycoated paper at 25× magnification. The fibrous material wasproduced on a rotary knife cutter utilizing a screen with ⅛ inchopenings.

FIG. 27 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was produced on a rotary knife cutter utilizing a screen with ⅛inch openings.

FIG. 28 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was twice sheared on a rotary knife cutter utilizing a screenwith 1/16 inch openings during each shearing.

FIG. 29 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was thrice sheared on a rotary knife cutter. During the firstshearing, a ⅛ inch screen was used; during the second shearing, a 1/16inch screen was used, and during the third shearing a 1/32 inch screenwas used.

FIGS. 29A-29F are 3-D Raman spectra from the surface of fibers fromsamples P132, P132-10, P132-100, P-1e, P-30e, and P-100e, respectively.

FIG. 30 is a schematic side view of a sonication apparatus, while FIG.31 is a cross-sectional view through the processing cell of FIG. 30.

FIG. 32 is a scanning electron micrograph at 1000× magnification of afibrous material produced from shearing switchgrass on a rotary knifecutter, and then passing the sheared material through a 1/32 inchscreen.

FIGS. 33 and 34 are scanning electron micrographs of the fibrousmaterial of FIG. 32 after irradiation with 10 Mrad and 100 Mrad gammarays, respectively, at 1000× magnification.

FIG. 35 is a scanning electron micrographs of the fibrous material ofFIG. 32 after irradiation with 10 Mrad and sonication at 1000×magnification.

FIG. 36 is a scanning electron micrographs of the fibrous material ofFIG. 32 after irradiation with 100 Mrad and sonication at 1000×magnification.

FIG. 37 is an infrared spectrum of Kraft board paper sheared on a rotaryknife cutter.

FIG. 38 is an infrared spectrum of the Kraft paper of FIG. 37 afterirradiation with 100 Mrad of gamma radiation.

FIGS. 38A-38I are ¹H-NMR spectra of samples P132, P132-10, P132-100,P-1e, P-5e, P-10e, P-30e, P-70e, and P-100e in Example 23. FIG. 38J is acomparison of the exchangeable proton at 16 ppm from FIGS. 38A-38I. FIG.38K is a ¹³C-NMR of sample P-100e. FIGS. 38L-38M are ¹³C-NMR of sampleP-100e with a delay time of 10 seconds. FIG. 38N is a 1H-NMR at aconcentration of 10% wt./wt. of sample P-100e.

FIG. 39 shows glucose concentration vs. time for various feedstocks.

FIGS. 40A, 40B, and 40C show cell concentrations, ethanolconcentrations, and percent growth and ethanol production, respectively,for Z. mobilis.

FIGS. 41A, 41B, and 41C show cell concentrations, ethanolconcentrations, and percent growth and ethanol production, respectively,for P. stipitis.

FIGS. 42A, 42B, and 42C show cell concentrations, ethanolconcentrations, and percent growth and ethanol production, respectively,for S. cerevisiae.

FIG. 43 is a schematic view of a process for biomass conversion.

FIG. 44 is schematic view of another process for biomass conversion.

DETAILED DESCRIPTION

Biomass (e.g., plant biomass, such as those that are or that include oneor more low molecular weight sugars, animal biomass, and municipal wastebiomass) can be processed to produce useful products such as fuels,e.g., fuels for internal combustion engines, jet engines or feedstocksfor fuel cells. In addition, functionalized materials having desiredtypes and amounts of functionality, such as carboxylic acid groups, enolgroups, aldehyde groups, ketone groups, nitrile groups, nitro groups, ornitroso groups, can be prepared using the methods described herein. Suchfunctionalized materials can be, e.g., more soluble, easier to utilizeby various microorganisms or can be more stable over the long term,e.g., less prone to oxidation. Systems and processes are describedherein that can use various biomass materials, such as cellulosicmaterials, lignocellulosic materials, starchy materials or materialsthat are or that include low molecular weight sugars, as feedstockmaterials. Such materials are often readily available, but can bedifficult to process, e.g., by fermentation, or can give sub-optimalyields at a slow rate.

Feedstock materials are first physically prepared for processing, oftenby size reduction of raw feedstock materials. Physically preparedfeedstock can be pretreated or processed using one or more of radiation,sonication, oxidation, pyrolysis, and steam explosion.

The various pretreatment systems and methods can be used in combinationsof two, three, or even four of these technologies.

In some cases, to provide materials that include a carbohydrate, such ascellulose, that can be converted by a microorganism to a number ofdesirable products, such as a combustible fuels (e.g., ethanol, butanolor hydrogen), feedstocks that include one or more saccharide units canbe treated by any one or more of the processes described herein. Otherproducts and co-products that can be produced include, for example,human food, animal feed, pharmaceuticals, and nutriceuticals. A numberof examples are presented that range from bench scale implementations ofindividual pretreatment methods to large-scale biomass processingplants.

Types of Biomass

Generally, any biomass material that is or includes carbohydratescomposed entirely of one or more saccharide units or that include one ormore saccharide units can be processed by any of the methods describedherein. For example, the biomass material can be cellulosic orlignocellulosic materials, starchy materials, such as kernels of corn,grains of rice or other foods, or materials that are or that include oneor more low molecular weight sugars, such as sucrose or cellobiose.

For example, such materials can include paper, paper products, wood,wood-related materials, particle board, grasses, rice hulls, bagasse,cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, ricehulls, coconut hair, algae, seaweed, cotton, synthetic celluloses, ormixtures of any of these. Suitable materials include those listed in theSummary section, above.

Fiber sources include cellulosic fiber sources, including paper andpaper products (e.g., polycoated paper and Kraft paper), andlignocellulosic fiber sources, including wood, and wood-relatedmaterials, e.g., particleboard. Other suitable fiber sources includenatural fiber sources, e.g., grasses, rice hulls, bagasse, cotton, jute,hemp, flax, bamboo, sisal, abaca, straw, corn cobs, rice hulls, coconuthair; fiber sources high in a-cellulose content, e.g., cotton; andsynthetic fiber sources, e.g., extruded yarn (oriented yarn orun-oriented yarn). Natural or synthetic fiber sources can be obtainedfrom virgin scrap textile materials, e.g., remnants or they can bepost-consumer waste, e.g., rags. When paper products are used as fibersources, they can be virgin materials, e.g., scrap virgin materials, orthey can be post-consumer waste. Aside from virgin raw materials,post-consumer, industrial (e.g., offal), and processing waste (e.g.,effluent from paper processing) can also be used as fiber sources. Also,the fiber source can be obtained or derived from human (e.g., sewage),animal or plant wastes. Additional fiber sources have been described inU.S. Pat. Nos. 6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105.

In some embodiments, the carbohydrate is or includes a material havingone or more -1,4-linkages and having a number average molecular weightbetween about 3,000 and 50,000. Such a carbohydrate is or includescellulose (I), which is derived from (β-glucose 1) through condensationof (1-4)-glycosidic bonds. This linkage contrasts itself with that for a(1-4)-glycosidic bonds present in starch and other carbohydrates.

Starchy materials include starch itself, e.g., corn starch, wheatstarch, potato starch or rice starch, a derivative of starch, or amaterial that includes starch, such as an edible food product or a crop.For example, the starchy material can be arracacha, buckwheat, banana,barley, cassava, kudzu, ocra, sago, sorghum, regular household potatoes,sweet potato, taro, yarns, or one or more beans, such as favas, lentilsor peas.

Blends of these and/or other starchy materials are also considered to bestarchy materials. In particular embodiments, the starchy material isderived from corn. Various corn starches and derivatives are describedin “Corn Starch,” Corn Refiners Association (11^(th) Edition, 2006),which is attached hereto as Appendix A.

Biomass materials that include low molecular weight sugars can, e.g.,include at least about 0.5 percent by weight of the low molecular sugar,e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 25, 35, 50, 60,70, 80, 90 or even at least about 95 percent by weight of the lowmolecular weight sugar. In some instances, the biomass is composedsubstantially of the low molecular weight sugar, e.g., greater than 95percent by weight, such as 96, 97, 98, 99 or substantially 100 percentby weight of the low molecular weight sugar.

Biomass materials that include low molecular weight sugars can beagricultural products or food products, such as sugarcane and sugarbeets, or an extract therefrom, e.g., juice from sugarcane or sugarbeets. Biomass materials that include low molecular weight sugars can besubstantially pure extracts, such as raw or crystallized table sugar(sucrose). Low molecular weight sugars include sugar derivatives. Forexample, the low molecular weight sugars can be oligomeric (e.g., equalto or greater than a 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer or10-mer), trimeric, dimeric, or monomeric. When the carbohydrates areformed of more than a single repeat unit, each repeat unit can be thesame or different.

Specific examples of low molecular weight sugars include cellobiose,lactose, sucrose, glucose and xylose, along with derivatives thereof. Insome instances, sugar derivatives are more rapidly dissolved in solutionor utilized by microbes to provide a useful material, such as ethanol orbutanol. Several such sugars and sugar derivatives are shown below.

Blends of any biomass materials described herein can be utilized formaking any of the products described herein, such as ethanol. Forexample, blends of cellulosic materials and starchy materials can beutilized for making any product described herein.

Systems for Treating Biomass

FIG. 1 shows a system 100 for converting biomass, particularly biomasswith significant cellulosic and lignocellulosic components and/orstarchy components, into useful products and co-products. System 100includes a feed preparation subsystem 110, a pretreatment subsystem 114,a primary process subsystem 118, and a post-processing subsystem 122.Feed preparation subsystem 110 receives biomass in its raw form,physically prepares the biomass for use as feedstock by downstreamprocesses (e.g., reduces the size of and homogenizes the biomass), andstores the biomass both in its raw and feedstock forms. Biomassfeedstock with significant cellulosic and/or lignocellulosic components,or starchy components can have a high average molecular weight andcrystallinity that can make processing the feedstock into usefulproducts (e.g., fermenting the feedstock to produce ethanol) difficult.For example, others have used acids, bases and enzymes to processcellulosic, lignocellulosic or starchy feedstocks. As described herein,in some embodiments, such treatments are unnecessary, or are necessaryonly in small or catalytic amounts.

Pretreatment subsystem 114 receives feedstock from the feed preparationsubsystem 110 and prepares the feedstock for use in primary productionprocesses by, for example, reducing the average molecular weight andcrystallinity of the feedstock. Primary process subsystem 118 receivespretreated feedstock from pretreatment subsystem 114 and produces usefulproducts (e.g., ethanol, other alcohols, pharmaceuticals, and/or foodproducts). In some cases, the output of primary process subsystem 118 isdirectly useful but, in other cases, requires further processingprovided by post-processing subsystem 122. Post-processing subsystem 122provides further processing to product streams from primary processsystem 118 which require it (e.g., distillation and denaturation ofethanol) as well as treatment for waste streams from the othersubsystems. In some cases, the co-products of subsystems 114, 118, 122can also be directly or indirectly useful as secondary products and/orin increasing the overall efficiency of system 100. For example,post-processing subsystem 122 can produce treated water to be recycledfor use as process water in other subsystems and/or can produce burnablewaste which can be used as fuel for boilers producing steam and/orelectricity.

The optimum size for biomass conversion plants is affected by factorsincluding economies of scale and the type and availability of biomassused as feedstock. Increasing plant size tends to increase economies ofscale associated with plant processes. However, increasing plant sizealso tends to increase the costs (e.g., transportation costs) per unitof feedstock. Studies analyzing these factors suggest that theappropriate size for biomass conversion plants can range from 2000 to10,000 dried tons of feedstock per day depending at least in part on thetype of feedstock used. The type of feedstock can also impact plantstorage requirements with plants designed primarily for processingfeedstock whose availability varies seasonally (e.g., corn stover)requiring more on- or off-site feedstock storage than plants designed toprocess feedstock whose availability is relatively steady (e.g., wastepaper).

Physical Preparation

In some cases, methods of processing begin with a physical preparationof the feedstock, e.g., size reduction of raw feedstock materials, suchas by cutting, grinding, shearing or chopping. In some cases, loosefeedstock (e.g., recycled paper, starchy materials, or switchgrass) isprepared by shearing or shredding. Screens and/or magnets can be used toremove oversized or undesirable objects such as, for example, rocks ornails from the feed stream.

Feed preparation systems can be configured to produce feed streams withspecific characteristics such as, for example, specific maximum sizes,specific length-to-width, or specific surface areas ratios. As a part offeed preparation, the bulk density of feedstocks can be controlled(e.g., increased or decreased).

Size Reduction

In some embodiments, the material to be processed is in the form of afibrous material that includes fibers provided by shearing a fibersource. For example, the shearing can be performed with a rotary knifecutter.

For example, and by reference to FIG. 2, a fiber source 210 is sheared,e.g., in a rotary knife cutter, to provide a first fibrous material 212.The first fibrous material 212 is passed through a first screen 214having an average opening size of 1.59 mm or less ( 1/16 inch, 0.0625inch) to provide a second fibrous material 216. If desired, fiber sourcecan be cut prior to the shearing, e.g., with a shredder. For example,when a paper is used as the fiber source, the paper can be first cutinto strips that are, e.g., ¼- to ½-inch wide, using a shredder, e.g., acounter-rotating screw shredder, such as those manufactured by Munson(Utica, N.Y.). As an alternative to shredding, the paper can be reducedin size by cutting to a desired size using a guillotine cutter. Forexample, the guillotine cutter can be used to cut the paper into sheetsthat are, e.g., 10 inches wide by 12 inches long.

In some embodiments, the shearing of fiber source and the passing of theresulting first fibrous material through first screen are performedconcurrently. The shearing and the passing can also be performed in abatch-type process.

For example, a rotary knife cutter can be used to concurrently shear thefiber source and screen the first fibrous material. Referring to FIG. 3,a rotary knife cutter 220 includes a hopper 222 that can be loaded witha shredded fiber source 224 prepared by shredding fiber source. Shreddedfiber source is sheared between stationary blades 230 and rotatingblades 232 to provide a first fibrous material 240. First fibrousmaterial 240 passes through screen 242, and the resulting second fibrousmaterial 244 is captured in bin 250. To aid in the collection of thesecond fibrous material, the bin can have a pressure below nominalatmospheric pressure, e.g., at least 10 percent below nominalatmospheric pressure, e.g., at least 25 percent below nominalatmospheric pressure, at least 50 percent below nominal atmosphericpressure, or at least 75 percent below nominal atmospheric pressure. Insome embodiments, a vacuum source 252 is utilized to maintain the binbelow nominal atmospheric pressure.

Shearing can be advantageous for “opening up” and “stressing” thefibrous materials, making the cellulose of the materials moresusceptible to chain scission and/or reduction of crystallinity. Theopen materials can also be more susceptible to oxidation whenirradiated.

The fiber source can be sheared in a dry state, a hydrated state (e.g.,having up to ten percent by weight absorbed water), or in a wet state,e.g., having between about 10 percent and about 75 percent by weightwater. The fiber source can even be sheared while partially or fullysubmerged under a liquid, such as water, ethanol, isopropanol.

The fiber source can also be sheared in under a gas (such as a stream oratmosphere of gas other than air), e.g., oxygen or nitrogen, or steam.

Other methods of making the fibrous materials include, e.g., stonegrinding, mechanical ripping or tearing, pin grinding or air attritionmilling.

If desired, the fibrous materials can be separated, e.g., continuouslyor in batches, into fractions according to their length, width, density,material type, or some combination of these attributes.

For example, ferrous materials can be separated from any of the fibrousmaterials by passing a fibrous material that includes a ferrous materialpast a magnet, e.g., an electromagnet, and then passing the resultingfibrous material through a series of screens, each screen havingdifferent sized apertures.

The fibrous materials can also be separated, e.g., by using a highvelocity gas, e.g., air. In such an approach, the fibrous materials areseparated by drawing off different fractions, which can be characterizedphotonically, if desired. Such a separation apparatus is discussed inLindsey et al, U.S. Pat. No. 6,883,667.

The fibrous materials can irradiated immediately following theirpreparation, or they can may be dried, e.g., at approximately 105° C.for 4-18 hours, so that the moisture content is, e.g., less than about0.5% before use.

If desired, lignin can be removed from any of the fibrous materials thatinclude lignin. Also, to aid in the breakdown of the materials thatinclude the cellulose, the material can be treated prior to irradiationwith heat, a chemical (e.g., mineral acid, base or a strong oxidizersuch as sodium hypochlorite) and/or an enzyme.

In some embodiments, the average opening size of the first screen isless than 0.79 mm ( 1/32 inch, 0.03125 inch), e.g., less than 0.51 mm (1/50 inch, 0.02000 inch), less than 0.40 mm ( 1/64 inch, 0.015625 inch),less than 0.23 mm (0.009 inch), less than 0.20 mm ( 1/128 inch,0.0078125 inch), less than 0.18 mm (0.007 inch), less than 0.13 mm(0.005 inch), or even less than less than 0.10 mm ( 1/256 inch,0.00390625 inch). The screen is prepared by interweaving monofilamentshaving an appropriate diameter to give the desired opening size. Forexample, the monofilaments can be made of a metal, e.g., stainlesssteel. As the opening sizes get smaller, structural demands on themonofilaments may become greater. For example, for opening sizes lessthan 0.40 mm, it can be advantageous to make the screens frommonofilaments made from a material other than stainless steel, e.g.,titanium, titanium alloys, amorphous metals, nickel, tungsten, rhodium,rhenium, ceramics, or glass. In some embodiments, the screen is madefrom a plate, e.g., a metal plate, having apertures, e.g., cut into theplate using a laser. In some embodiments, the open area of the mesh isless than 52%, e.g., less than 41%, less than 36%, less than 31%, lessthan 30%.

In some embodiments, the second fibrous is sheared and passed throughthe first screen, or a different sized screen. In some embodiments, thesecond fibrous material is passed through a second screen having anaverage opening size equal to or less than that of first screen.

Referring to FIG. 4, a third fibrous material 220 can be prepared fromthe second fibrous material 216 by shearing the second fibrous material216 and passing the resulting material through a second screen 222having an average opening size less than the first screen 214.

Generally, the fibers of the fibrous materials can have a relativelylarge average length-to-diameter ratio (e.g., greater than 20-to-1),even if they have been sheared more than once. In addition, the fibersof the fibrous materials described herein may have a relatively narrowlength and/or length-to-diameter ratio distribution.

As used herein, average fiber widths (i.e., diameters) are thosedetermined optically by randomly selecting approximately 5,000 fibers.Average fiber lengths are corrected length-weighted lengths. BET(Brunauer, Emmet and Teller) surface areas are multi-point surfaceareas, and porosities are those determined by mercury porosimetry.

The average length-to-diameter ratio of the second fibrous material 14can be, e.g., greater than 8/1, e.g., greater than 10/1, greater than15/1, greater than 20/1, greater than 25/1, or greater than 50/1. Anaverage length of the second fibrous material 14 can be, e.g., betweenabout 0.5 mm and 2.5 mm, e.g., between about 0.75 mm and 1.0 mm, and anaverage width (i.e., diameter) of the second fibrous material 14 can be,e.g., between about 5 μm and 50 μm, e.g., between about 10 μm and 30 μm.

In some embodiments, a standard deviation of the length of the secondfibrous material 14 is less than 60 percent of an average length of thesecond fibrous material 14, e.g., less than 50 percent of the averagelength, less than 40 percent of the average length, less than 25 percentof the average length, less than 10 percent of the average length, lessthan 5 percent of the average length, or even less than 1 percent of theaverage length.

In some embodiments, a BET surface area of the second fibrous materialis greater than 0.1 m²/g, e.g., greater than 0.25 m²/g, greater than 0.5m²/g, greater than 1.0 m²/g, greater than 1.5 m²/g, greater than 1.75m²/g, greater than 5.0 m²/g, greater than 10 m²/g, greater than 25 m²/g,greater than 35 m²/g, greater than 50 m²/g, greater than 60 m²/g,greater than 75 m²/g, greater than 100 m²/g, greater than 150 m²/g,greater than 200 m²/g, or even greater than 250 m²/g. A porosity of thesecond fibrous material 14 can be, e.g., greater than 20 percent,greater than 25 percent, greater than 35 percent, greater than 50percent, greater than 60 percent, greater than 70 percent, e.g., greaterthan 80 percent, greater than 85 percent, greater than 90 percent,greater than 92 percent, greater than 94 percent, greater than 95percent, greater than 97.5 percent, greater than 99 percent, or evengreater than 99.5 percent.

In some embodiments, a ratio of the average length-to-diameter ratio ofthe first fibrous material to the average length-to-diameter ratio ofthe second fibrous material is, e.g., less than 1.5, e.g., less than1.4, less than 1.25, less than 1.1, less than 1.075, less than 1.05,less than 1.025, or even substantially equal to 1.

In particular embodiments, the second fibrous material is sheared againand the resulting fibrous material passed through a second screen havingan average opening size less than the first screen to provide a thirdfibrous material. In such instances, a ratio of the averagelength-to-diameter ratio of the second fibrous material to the averagelength-to-diameter ratio of the third fibrous material can be, e.g.,less than 1.5, e.g., less than 1.4, less than 1.25, or even less than1.1.

In some embodiments, the third fibrous material is passed through athird screen to produce a fourth fibrous material. The fourth fibrousmaterial can be, e.g., passed through a fourth screen to produce a fifthmaterial. Similar screening processes can be repeated as many times asdesired to produce the desired fibrous material having the desiredproperties.

Densification

Densified materials can be processed by any of the methods describedherein, or any material described herein, e.g., any fibrous materialdescribed herein, can be processed by any one or more methods describedherein, and then densified as described herein.

A material, e.g., a fibrous material, having a low bulk density can bedensified to a product having a higher bulk density. For example, amaterial composition having a bulk density of 0.05 g/cm³ can bedensified by sealing the fibrous material in a relatively gasimpermeable structure, e.g., a bag made of polyethylene or a bag made ofalternating layers of polyethylene and a nylon, and then evacuating theentrapped gas, e.g., air, from the structure. After evacuation of theair from the structure, the fibrous material can have, e.g., a bulkdensity of greater than 0.3 g/cm³, e.g., 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³or more, e.g., 0.85 g/cm³. After densification, the product canprocessed by any of the methods described herein, e.g., irradiated,e.g., with gamma radiation. This can be advantageous when it isdesirable to transport the material to another location, e.g., a remotemanufacturing plant, where the fibrous material composition can be addedto a solution, e.g., to produce ethanol. After piercing thesubstantially gas impermeable structure, the densified fibrous materialcan revert to nearly its initial bulk density, e.g., greater than 60percent of its initial bulk density, e.g., 70 percent, 80 percent, 85percent or more, e.g., 95 percent of its initial bulk density. To reducestatic electricity in the fibrous material, an antistatic agent can beadded to the material.

In some embodiments, the structure, e.g., bag, is formed of a materialthat dissolves in a liquid, such as water. For example, the structurecan be formed from a polyvinyl alcohol so that it dissolves when incontact with a water-based system. Such embodiments allow densifiedstructures to be added directly to solutions that include amicroorganism, without first releasing the contents of the structure,e.g., by cutting.

Referring to FIG. 5, a biomass material can be combined with any desiredadditives and a binder, and subsequently densified by application ofpressure, e.g., by passing the material through a nip defined betweencounter-rotating pressure rolls or by passing the material through apellet mill. During the application of pressure, heat can optionally beapplied to aid in the densification of the fibrous material. Thedensified material can then be irradiated.

In some embodiments, the material prior to densification has a bulkdensity of less than 0.25 g/cm3, e.g., 0.20 g/cm3, 0.15 g/cm3, 0.10g/cm3, 0.05 g/cm3 or less, e.g., 0.025 g/cm3. Bulk density is determinedusing ASTM D1895B. Briefly, the method involves filling a measuringcylinder of known volume with a sample and obtaining a weight of thesample. The bulk density is calculated by dividing the weight of thesample in grams by the known volume of the cylinder in cubiccentimeters.

The preferred binders include binders that are soluble in water, swollenby water, or that has a glass transition temperature of less 25° C., asdetermined by differential scanning calorimetry. By water-solublebinders, we mean binders having a solubility of at least about 0.05weight percent in water. By water soluble binders, we mean binders thatincrease in volume by more than 0.5 percent upon exposure to water.

In some embodiments, the binders that are soluble or swollen by waterinclude a functional group that is capable of forming a bond, e.g., ahydrogen bond, with the fibers of the fibrous material, e.g., cellulosicfibrous material. For example, the functional group can be a carboxylicacid group, a carboxylate group, a carbonyl group, e.g., of an aldehydeor a ketone, a sulfonic acid group, a sulfonate group, a phosphoric acidgroup, a phosphate group, an amide group, an amine group, a hydroxylgroup, e.g., of an alcohol, and combinations of these groups, e.g., acarboxylic acid group and a hydroxyl group. Specific monomeric examplesinclude glycerin, glyoxal, ascorbic acid, urea, glycine,pentaerythritol, a monosaccharide or a disaccharide, citric acid, andtartaric acid. Suitable saccharides include glucose, sucrose, lactose,ribose, fructose, mannose, arabinose and erythrose. Polymeric examplesinclude polyglycols, polyethylene oxide, polycarboxylic acids,polyamides, polyamines and polysulfonic acids polysulfonates. Specificpolymeric examples include polypropylene glycol (PPG), polyethyleneglycol (PEG), polyethylene oxide, e.g., POLYOX®, copolymers of ethyleneoxide and propylene oxide, polyacrylic acid (PAA), polyacrylamide,polypeptides, polyethylenimine, polyvinylpyridine,poly(sodium-4-styrenesulfonate) andpoly(2-acrylamido-methyl-1-propanesulfonic acid).

In some embodiments, the binder includes a polymer that has a glasstransition temperature less 25° C. Examples of such polymers includethermoplastic elastomers (TPEs). Examples of TPEs include polyetherblock amides, such as those available under the trade name PEBAX®,polyester elastomers, such as those available under the trade nameHYTREL®′ and styrenic block copolymers, such as those available underthe trade name KRATON®. Other suitable polymers having a glasstransition temperature less 25° C. include ethylene vinyl acetatecopolymer (EVA), polyolefins, e.g., polyethylene, polypropylene,ethylene-propylene copolymers, and copolymers of ethylene and alphaolefins, e.g., 1-octene, such as those available under the trade name

ENGAGE®. In some embodiments, e.g., when the material is a fiberizedpolycoated paper, the material is densified without the addition of aseparate low glass transition temperature polymer.

In a particular embodiment, the binder is a lignin, e.g., a natural orsynthetically modified lignin.

A suitable amount of binder added to the material, calculated on a dryweight basis, is, e.g., from about 0.01 percent to about 50 percent,e.g., 0.03 percent, 0.05 percent, 0.1 percent, 0.25 percent, 0.5percent, 1.0 percent, 5 percent, 10 percent or more, e.g., 25 percent,based on a total weight of the densified material. The binder can beadded to the material as a neat, pure liquid, as a liquid having thebinder dissolved therein, as a dry powder of the binder, or as pelletsof the binder.

The densified fibrous material can be made in a pellet mill. Referringto FIG. 6, a pellet mill 300 has a hopper 301 for holding undensifiedmaterial 310 that includes a carbohydrate-containing material, such ascellulose. The hopper communicates with an auger 312 that is driven byvariable speed motor 314 so that undensified material can be transportedto a conditioner 320 that stirs the undensified material with paddles322 that are rotated by conditioner motor 330. Other ingredients, e.g.,any of the additives and/or fillers described herein, can be added atinlet 332. If desired, heat may be added while the fibrous material isin conditioner. After conditioned, the material passes from theconditioner through a dump chute 340, and to another auger 342. The dumpchute, as controlled by actuator 344, allows for unobstructed passage ofthe material from conditioner to auger. Auger is rotated by motor 346,and controls the feeding of the fibrous material into die and rollerassembly 350. Specifically, the material is introduced into a hollow,cylindrical die 352, which rotates about a horizontal axis and which hasradially extending die holes 250. Die 352 is rotated about the axis bymotor 360, which includes a horsepower gauge, indicating total powerconsumed by the motor. Densified material 370, e.g., in the form ofpellets, drops from chute 372 and are captured and processed, such as byirradiation.

The material, after densification, can be conveniently in the form ofpellets or chips having a variety of shapes. The pellets can then beirradiated. In some embodiments, the pellets or chips are cylindrical inshape, e.g., having a maximum

transverse dimension of, e.g., 1 mm or more, e.g., 2 mm, 3 mm, 5 mm, 8mm, 10 mm, 15 mm or more, e.g., 25 mm. Other convenient shapes includepellets or chips that are plate-like in form, e.g., having a thicknessof 1 mm or more, e.g., 2 mm, 3 mm, 5 mm, 8 mm, 10 mm or more, e.g., 25mm; a width of, e.g., 5 mm or more, e.g., 10 mm, 15 mm, 25 mm, 30 mm ormore, e.g., 50 mm; and a length of 5 mm or more, e.g., 10 mm, 15 mm, 25mm, 30 mm or more, e.g., 50 mm.

Referring now FIG. 7A-7D, pellets can be made so that they have a hollowinside. As shown, the hollow can be generally in-line with the center ofthe pellet (FIG. 7B), or out of line with the center of the pellet (FIG.7C). Making the pellet hollow inside can increase the rate ofdissolution in a liquid after irradiation.

Referring now to FIG. 7D, the pellet can have, e.g., a transverse shapethat is multi-lobal, e.g., tri-lobal as shown, or tetra-lobal,penta-lobal, hexa-lobal or deca-lobal. Making the pellets in suchtransverse shapes can also increase the rate of dissolution in asolution after irradiation.

Alternatively, the densified material can be in any other desired form,e.g., the densified material can be in the form of a mat, roll or bale.

Examples

In one example, half-gallon juice cartons made of un-printed white Kraftboard having a bulk density of 20 lb/ft can be used as a feedstock.Cartons can be folded flat and then fed into a shredder to produce aconfetti-like material having a width of between 0.1 inch and 0.5 inch,a length of between 0.25 inch and 1 inch and a thickness equivalent tothat of the starting material (about 0.075 inch). The confetti-likematerial can be fed to a rotary knife cutter, which shears theconfetti-like pieces, tearing the pieces apart and releasing fibrousmaterial.

In some cases, multiple shredder-shearer trains can be arranged inseries with output. In one embodiment, two shredder-shearer trains canbe arranged in series with output from the first shearer fed as input tothe second shredder. In another embodiment, three shredder-shearertrains can be arranged in series with output from the first shearer fedas input to the second shredder and output from the second shearer fedas input to the third shredder. Multiple passes through shredder-shearertrains are anticipated to increase decrease particle size and increaseoverall surface area within the feedstream.

In another example, fibrous material produced from shredding andshearing juice cartons can be treated to increase its bulk density. Insome cases, the fibrous material can be sprayed with water or a dilutestock solution of POLYOX™ WSR NIO (polyethylene oxide) prepared inwater. The wetted fibrous material can then be processed through apellet mill operating at room temperature. The pellet mill can increasethe bulk density of the feedstream by more than an order of magnitude.

Pretreatment

Physically prepared feedstock can be pretreated for use in primaryproduction processes by, for example, reducing the average molecularweight and crystallinity of the feedstock and/or increasing the surfacearea and/or porosity of the feedstock. In some embodiments, thecellulosic and/or lignocellulosic material includes a first cellulosehaving a first number average molecular weight and the resultingcarbohydrate includes a second cellulose having a second number averagemolecular weight lower than the first number average molecular weight.For example, the second number average molecular weight is lower thanthe first number average molecular weight by more than about twenty-fivepercent, e.g., 2×, 3×, 5×, 7×, 10×, 25×, even 100× reduction.

In some embodiments, the first cellulose has a first crystallinity andthe second cellulose has a second crystallinity lower than the firstcrystallinity, such as lower than about two, three, five, ten, fifteenor twenty-five percent lower.

In some embodiments, the first cellulose has a first level of oxidationand the second cellulose has a second level of oxidation higher than thefirst level of oxidation, such as two, three, four, five, ten or eventwenty-five percent higher.

Pretreatment processes can include one or more of irradiation,sonication, oxidation, pyrolysis, and steam explosion. The variouspretreatment systems and methods can be used in combinations of two,three, or even four of these technologies.

Pretreatment Combinations

In some embodiments, biomass can be processed by applying two or more ofany of the processes described herein, such as two, three, four or moreof radiation, sonication, oxidation, pyrolysis, and steam explosioneither with or without prior, intermediate, or subsequent feedstockpreparation as described herein. The processes can be applied to thebiomass in any order or concurrently. For example, a carbohydrate can beprepared by applying radiation, sonication, oxidation, pyrolysis, and,optionally, steam explosion to a cellulosic and/or lignocellulosicmaterial (in any order or concurrently).

The provided carbohydrate-containing material can then be converted byone or more microorganisms, such as bacteria, yeast, or mixtures ofyeast and bacteria, to a number of desirable products, as describedherein. Multiple processes can provide materials that can be morereadily utilized by a variety of microorganisms because of their lowermolecular weight, lower crystallinity, and/or enhanced solubility.Multiple processes can provide synergies and can reduce overall energyinput required in comparison to any single process.

For example, in some embodiments, feedstocks are provided that include acarbohydrate that is produced by a process that includes irradiating andsonicating, irradiating and oxidizing, irradiating and pyrolyzing, orirradiating and steam-exploding (in either order or concurrently) acellulosic and/or a lignocellulosic material. The provided feedstock canthen be contacted with a microorganism having the ability to convert atleast a portion, e.g., at least about 1 percent by weight, of thefeedstock to the product, such as the combustible fuel.

Pretreatment Conditions

In some embodiments, the process does not include hydrolyzing thecellulosic and/or lignocellulosic material, such as with an acid or abase, e.g., a mineral acid, such as hydrochloric or sulfuric acid. Ifdesired, some or none of the feedstock can include a hydrolyzedmaterial. For example, in some embodiments, at least about seventypercent by weight of the feedstock is an unhydrolyzed material, e.g., atleast at 95 percent by weight of the feedstock is an unhydrolyzedmaterial. In some embodiments, substantially all of the feedstock is anunhydrolyzed material.

Any feedstock or any reactor or fermentor charged with a feedstock caninclude a buffer, such as sodium bicarbonate, ammonium chloride or Tris;an electrolyte, such as potassium chloride, sodium chloride, or calciumchloride; a growth factor, such as biotin and/or a base pair such asuracil or an equivalent thereof; a surfactant, such as Tween® orpolyethylene glycol; a mineral, such as such as calcium, chromium,copper, iodine, iron, selenium, or zinc; or a chelating agent, such asethylene diamine, ethylene diamine tetraacetic acid (EDTA) (or its saltform, e.g., sodium or potassium EDTA), or dimercaprol.

When radiation is utilized, it can be applied to any sample that is dryor wet, or even dispersed in a liquid, such as water. For example,irradiation can be performed on cellulosic and/or lignocellulosicmaterial in which less than about 25 percent by weight of the cellulosicand/or lignocellulosic material has surfaces wetted with a liquid, suchas water. In some embodiments, irradiating is performed on cellulosicand/or lignocellulosic material in which substantially none of thecellulosic and/or lignocellulosic material is wetted with a liquid, suchas water.

In some embodiments, any processing described herein occurs after thecellulosic and/or lignocellulosic material remains dry as acquired orhas been dried, e.g., using heat and/or reduced pressure. For example,in some embodiments, the cellulosic and/or lignocellulosic material hasless than about five percent by weight retained water, measured at 25°C. and at fifty percent relative humidity.

If desired, a swelling agent, as defined herein, can be utilized in anyprocess described herein. In some embodiments, when a cellulosic and/orlignocellulosic material is processed using radiation, less than about25 percent by weight of the cellulosic and/or lignocellulosic materialis in a swollen state, the swollen state being characterized as having avolume of more than about 2.5 percent higher than an unswollen state,e.g., more than 5.0, 7.5, 10, or 15 percent higher than the unswollenstate. In some embodiments, when radiation is utilized on a cellulosicand/or lignocellulosic material, substantially none of the cellulosicand/or lignocellulosic material is in a swollen state. In specificembodiments when radiation is utilized, the cellulosic and/orlignocellulosic material includes a swelling agent, and swollencellulosic and/or lignocellulosic receives a dose of less than about 10Mrad.

When radiation is utilized in any process, it can be applied while thecellulosic and/or lignocellulosic is exposed to air, oxygen-enrichedair, or even oxygen itself, or blanketed by an inert gas such asnitrogen, argon, or helium. When maximum oxidation is desired, anoxidizing environment is utilized, such as air or oxygen.

When radiation is utilized, it may be applied to biomass, such ascellulosic and/or lignocellulosic material, under a pressure of greaterthan about 2.5 atmospheres, such as greater than 5, 10, 15, 20 or evengreater than about 50 atmospheres.

Radiation Treatment

One or more irradiation processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences. Irradiation can reduce the molecular weight and/orcrystallinity of feedstock. In some embodiments, energy deposited in amaterial that releases an electron from its atomic orbital is used toirradiate the materials. The radiation may be provided by 1) heavycharged particles, such as alpha particles or protons, 2) electrons,produced, for example, in beta decay or electron beam accelerators, or3) electromagnetic radiation, for example, gamma rays, x rays, orultraviolet rays. In one approach, radiation produced by radioactivesubstances can be used to irradiate the feedstock. In some embodiments,any combination in any order or concurrently of (1) through (3) may beutilized. In another approach, electromagnetic radiation (e.g., producedusing electron beam emitters) can be used to irradiate the feedstock.The doses applied depend on the desired effect and the particularfeedstock. For example, high doses of radiation can break chemical bondswithin feedstock components and low doses of radiation can increasechemical bonding (e.g., cross-linking) within feedstock components. Insome instances when chain scission is desirable and/or polymer chainfunctionalization is desirable, particles heavier than electrons, suchas protons, helium nuclei, argon ions, silicon ions, neon ions, carbonions, phosphorus ions, oxygen ions or nitrogen ions can be utilized.When ring-opening chain scission is desired, positively chargedparticles can be utilized for their Lewis acid properties for enhancedring-opening chain scission. For example, when oxygen-containingfunctional groups are desired, irradiation in the presence of oxygen oreven irradiation with oxygen ions can be performed. For example, whennitrogen-containing functional groups are desirable, irradiation in thepresence of nitrogen or even irradiation with nitrogen ions can beperformed.

Referring to FIG. 8, in one method, a first material that is or includeshaving a first number average molecular weight (TMN1) is irradiated,e.g., by treatment with ionizing radiation (e.g., in the form of gammaradiation, X-ray radiation, 100 nm to 280 nm ultraviolet (UV) light, abeam of electrons or other charged particles) to provide a secondmaterial 3 that includes cellulose having a second number averagemolecular weight (TMN2) lower than the first number average molecularweight. The second material (or the first and second material) can becombined with a microorganism (e.g., a bacterium or a yeast) that canutilize the second and/or first material to produce a fuel that is orincludes hydrogen, an alcohol (e.g., ethanol or butanol, such as n-,sec- or t-butanol), an organic acid, a hydrocarbon or mixtures of any ofthese.

Since the second material 3 has cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable and/or soluble in a solution containing amicroorganism. These properties make the second material 3 moresusceptible to chemical, enzymatic and/or biological attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Radiationcan also sterilize the materials.

In some embodiments, the second number average molecular weight (MN2) islower than the first number average molecular weight (TMN1) by more thanabout 10 percent, e.g., 15, 20, 25, 30, 35, 40, 50 percent, 60 percent,or even more than about 75 percent.

In some instances, the second material has cellulose that has acrystallinity (TC2) that is lower than the crystallinity (TC1) of thecellulose of the first material. For example, (TC2) can be lower than(TC1) by more than about 10 percent, e.g., 15, 20, 25, 30, 35, 40, oreven more than about 50 percent.

In some embodiments, the starting crystallinity index (prior toirradiation) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after irradiation is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in some embodiments, e.g., after extensiveirradiation, it is possible to have a crystallinity index of lower than5 percent. In some embodiments, the material after irradiation issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto irradiation) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after irradiation is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive irradiation, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(TO2) that is higher than the level of oxidation (TO1) of the firstmaterial. A higher level of oxidation of the material can aid in itsdispersibility, swellability and/or solubility, further enhancing thematerials susceptibility to chemical, enzymatic or biological attack. Insome embodiments, to increase the level of the oxidation of the secondmaterial relative to the first material, the irradiation is performedunder an oxidizing environment, e.g., under a blanket of air or oxygen,producing a second material that is more oxidized than the firstmaterial. For example, the second material can have more hydroxyl groupsaldehyde groups, ketone groups, ester groups or carboxylic acid groups,which can increase its hydrophilicity.

Ionizing Radiation

Each form of radiation ionizes the biomass via particular interactions,as determined by the energy of the radiation. Heavy charged particlesprimarily ionize matter via Coulomb scattering; furthermore, theseinteractions produce energetic electrons that may further ionize matter.Alpha particles are identical to the nucleus of a helium atom and areproduced by the alpha decay of various radioactive nuclei, such asisotopes of bismuth, polonium, astatine, radon, francium, radium,several actinides, such as actinium, thorium, uranium, neptunium,curium, californium, americium, and plutonium.

When particles are utilized, they can be neutral (uncharged), positivelycharged or negatively charged. When charged, the charged particles canbear a single positive or negative charge, or multiple charges, e.g.,one, two, three or even four or more charges. In instances in whichchain scission is desired, positively charged particles may bedesirable, in part, due to their acidic nature. When particles areutilized, the particles can have the mass of a resting electron, orgreater, e.g., 500, 1000, 1500, or 2000 or more times the mass of aresting electron. For example, the particles can have a mass of fromabout 1 atomic unit to about 150 atomic units, e.g., from about 1 atomicunit to about 50 atomic units, or from about 1 to about 25, e.g., 1, 2,3, 4, 5, 10, 12 or 15 amu. Accelerators used to accelerate the particlescan be electrostatic DC, electrodynamic DC, RF linear, magneticinduction linear or continuous wave. For example, cyclotron typeaccelerators are available from IBA, Belgium, such as the Rhodotron®system, while DC type accelerators are available from RDI, now IBAIndustrial, such as the Dynamitron®. Ions and ion accelerators arediscussed in Introductory Nuclear Physics, Kenneth S. Krane, John Wiley& Sons, Inc. (1988), Krsto Prelec, FIZIKA B 6 (1997) 4, 177-206, a copyof which is attached as Appendix B, Chu, William T., “Overview ofLight-Ion Beam Therapy”, Columbus-Ohio, ICRU-IAEA Meeting, 18-20 Mar.2006, a copy of which is attached hereto at Appendix C, Iwata, Y. etal., “Alternating-Phase-Focused IH-DTL for Heavy-Ion MedicalAccelerators”, Proceedings of EPAC 2006, Edinburgh, Scotland, a copy ofwhich is attached hereto as Appendix D and Leitner, C. M. et al.,“Status of the Superconducting ECR Ion Source Venus”, Proceedings ofEPAC 2000, Vienna, Austria, which is attached hereto as Appendix E.

Electrons interact via Coulomb scattering and bremsstrahlung radiationproduced by changes in the velocity of electrons. Electrons may beproduced by radioactive nuclei that undergo beta decay, such as isotopesof iodine, cesium, technetium, and iridium. Alternatively, an electrongun can be used as an electron source via thermionic emission.

Electromagnetic radiation interacts via three processes: photoelectricabsorption, Compton scattering, and pair production. The dominatinginteraction is determined by the energy of the incident radiation andthe atomic number of the material. The summation of interactionscontributing to the absorbed radiation in cellulosic material can beexpressed by the mass absorption coefficient.

Electromagnetic radiation is subclassified as gamma rays, x rays,ultraviolet rays, infrared rays, microwaves, or radio waves, dependingon its wavelength.

For example, gamma radiation can be employed to irradiate the materials.Referring to FIGS. 9 and 10 (an enlarged view of region R), a gammairradiator 10 includes gamma radiation sources 408, e.g., 6° C. pellets,a working table 14 for holding the materials to be irradiated andstorage 16, e.g., made of a plurality iron plates, all of which arehoused in a concrete containment chamber (vault) 20 that includes a mazeentranceway 22 beyond a lead-lined door 26. Storage 16 includes aplurality of channels 30, e.g., sixteen or more channels, allowing thegamma radiation sources to pass through storage on their way proximatethe working table.

In operation, the sample to be irradiated is placed on a working table.The irradiator is configured to deliver the desired dose rate andmonitoring equipment is connected to an experimental block 31. Theoperator then leaves the containment chamber, passing through the mazeentranceway and through the lead-lined door. The operator mans a controlpanel 32, instructing a computer 33 to lift the radiation sources 12into working position using cylinder 36 attached to a hydraulic pump 40.

Gamma radiation has the advantage of a significant penetration depthinto a variety of material in the sample. Sources of gamma rays includeradioactive nuclei, such as isotopes of cobalt, calcium, technicium,chromium, gallium, indium, iodine, iron, krypton, samarium, selenium,sodium, thalium, and xenon.

Sources of x rays include electron beam collision with metal targets,such as tungsten or molybdenum or alloys, or compact light sources, suchas those produced commercially by Lyncean.

Sources for ultraviolet radiation include deuterium or cadmium lamps.

Sources for infrared radiation include sapphire, zinc, or selenidewindow ceramic lamps.

Sources for microwaves include klystrons, Slevin type RF sources, oratom beam sources that employ hydrogen, oxygen, or nitrogen gases.

Various other irradiating devices may be used in the methods disclosedherein,

including field ionization sources, electrostatic ion separators, fieldionization generators, thermionic emission sources, microwave dischargeion sources, recirculating or static accelerators, dynamic linearaccelerators, van de Graaff accelerators, and folded tandemaccelerators. Such devices are disclosed, for example, in U.S.Provisional Application Ser. No. 61/073,665, the complete disclosure ofwhich is incorporated herein by reference.

Electron Beam

In some embodiments, a beam of electrons is used as the radiationsource. A beam of electrons has the advantages of high dose rates (e.g.,1, 5, or even 10 Mrad per second), high throughput, less containment,and less confinement equipment. Electrons can also be more efficient atcausing chain scission. In addition, electrons having energies of 4-10MeV can have a penetration depth of 5 to 30 mm or more, such as 40 mm.

Electron beams can be generated, e.g., by electrostatic generators,cascade generators, transformer generators, low energy accelerators witha scanning system, low energy accelerators with a linear cathode, linearaccelerators, and pulsed accelerators. Electrons as an ionizingradiation source can be useful, e.g., for relatively thin piles ofmaterials, e.g., less than 0.5 inch, e.g., less than 0.4 inch, 0.3 inch,0.2 inch, or less than 0.1 inch. In some embodiments, the energy of eachelectron of the electron beam is from about 0.3 MeV to about 2.0 MeV(million electron volts), e.g., from about 0.5 MeV to about 1.5 MeV, orfrom about 0.7 MeV to about 1.25 MeV.

FIG. 11A shows a process flow diagram 3000 that includes various stepsin an electron beam irradiation feedstock pretreatment sequence. Infirst step 3010, a supply of dry feedstock is received from a feedsource. As discussed above, the dry feedstock from the feed source maybe pre-processed prior to delivery to the electron beam irradiationdevices. For example, if the feedstock is derived from plant sources,certain portions of the plant material may be removed prior tocollection of the plant material and/or before the plant material isdelivered by the feedstock transport device. Alternatively, or inaddition, as expressed in optional step 3020, the biomass feedstock canbe subjected to mechanical processing (e.g., to reduce the averagelength of fibers in the feedstock) prior to delivery to the electronbeam irradiation devices.

In step 3030, the dry feedstock is transferred to a feedstock transportdevice (e.g., a conveyor belt) and is distributed over thecross-sectional width of the feedstock transport device approximatelyuniformly by volume. This can be accomplished, for example, manually orby inducing a localized vibration motion at some point in the feedstocktransport device prior to the electron beam irradiation processing. Insome embodiments, a mixing system introduces a chemical agent 3045 intothe feedstock in an optional process 3040 that produces a slurry.Combining water with the processed feedstock in mixing step 3040 createsan aqueous feedstock slurry that may be transported through, forexample, piping rather than using, for example, a conveyor belt.

The next step 3050 is a loop that encompasses exposing the feedstock (indry or slurry form) to electron beam radiation via one or more (say, N)electron beam irradiation devices. The feedstock slurry is moved througheach of the N “showers” of electron beams at step 3052. The movement mayeither be at a continuous speed through and between the showers, orthere may be a pause through each shower, followed by a sudden movementto the next shower. A small slice of the feedstock slurry is exposed toeach shower for some predetermined exposure time at step 3053.

Electron beam irradiation devices may be procured commercially from IonBeam Applications, Louvain-la-Neuve, Belgium or the Titan Corporation,San Diego, Calif. Typical electron energies can be 1 MeV, 2 MeV, 4.5MeV, 7.5 MeV, or 10 MeV. Typical electron beam irradiation device powercan be 1 kW, 5 kW, 10 kW, 20 kW, 50 kW, 100 kW, 250 kW, or 500 kW.Effectiveness of depolymerization of the feedstock slurry depends on theelectron energy used and the dose applied, while exposure time dependson the power and dose. Typical doses may take values of 1 kGy, 5 kGy, 10kGy, 20 kGy, 50 kGy, 100 kGy, or 200 kGy.

Tradeoffs in considering electron beam irradiation device powerspecifications include cost to operate, capital costs, depreciation, anddevice footprint. Tradeoffs in considering exposure dose levels ofelectron beam irradiation would be energy costs and environment, safety,and health (ESH) concerns. Tradeoffs in considering electron energiesinclude energy costs; here, a lower electron energy may be advantageousin encouraging depolymerization of certain feedstock slurry (see, forexample, Bouchard, et al, Cellulose (2006) 13: 601-610). Typically,generators are housed in a vault, e.g., of lead or concrete orlead-lined concrete.

It may be advantageous to provide a double-pass of electron beamirradiation in order to provide a more effective depolymerizationprocess. For example, the feedstock transport device could direct thefeedstock (in dry or slurry form) underneath and in a reverse directionto its initial transport direction. Double-pass systems can allowthicker feedstock slurries to be processed and can provide a moreuniform depolymerization through the thickness of the feedstock slurry.

The electron beam irradiation device can produce either a fixed beam ora scanning beam. A scanning beam may be advantageous with large scansweep length and high scan speeds, as this would effectively replace alarge, fixed beam width. Further, available sweep widths of 0.5 m, 1 m,2 m or more are available.

Once a portion of feedstock slurry has been transported through the Nelectron beam irradiation devices, it may be necessary in someembodiments, as in step 3060, to mechanically separate the liquid andsolid components of the feedstock slurry. In these embodiments, a liquidportion of the feedstock slurry is filtered for residual solid particlesand recycled back to the slurry preparation step 3040. A solid portionof the feedstock slurry is then advanced on to the next processing step3070 via the feedstock transport device. In other embodiments, thefeedstock is maintained in slurry form for further processing.

Electromagnetic Radiation

In embodiments in which the irradiating is performed withelectromagnetic radiation, the electromagnetic radiation can have, e.g.,energy per photon (in electron volts) of greater than 10² eV, e.g.,greater than 10³, 10⁴, 10⁵, 10⁶, or even greater than 10⁷ eV. In someembodiments, the electromagnetic radiation has energy per photon ofbetween 10⁴ and 10⁷, e.g., between 10⁵ and 10⁶ eV. The electromagneticradiation can have a frequency of, e.g., greater than 10¹⁶ Hz, greaterthan 10¹⁷ Hz, 10¹⁸, 10¹⁹, 10²⁰, greater than 10²¹ Hz. In someembodiments, the electromagnetic radiation has a frequency of between10¹⁸ and 10²² Hz, e.g., between 10¹⁹ to 10²¹ Hz.

Doses

In some embodiments, the irradiating (with any radiation source or acombination of sources) is performed until the material receives a doseof at least 0.25 Mrad, e.g., at least 1.0 Mrad, at least 2.5 Mrad, atleast 5.0 Mrad, or at least 10.0 Mrad. In some embodiments, theirradiating is performed until the material receives a dose of between1.0 Mrad and 6.0 Mrad, e.g., between 1.5 Mrad and 4.0 Mrad.

In some embodiments, the irradiating is performed at a dose rate ofbetween 5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0kilorads/hour or between 50.0 and 350.0 kilorads/hours.

In some embodiments, two or more radiation sources are used, such as twoor more ionizing radiations. For example, samples can be treated, in anyorder, with a beam of electrons, followed by gamma radiation and UVlight having wavelengths from about 100 nm to about 280 nm. In someembodiments, samples are treated with three ionizing radiation sources,such as a beam of electrons, gamma radiation, and energetic UV light.

Alternatively, in another example, a fibrous material that includes acellulosic and/or lignocellulosic material is irradiated and,optionally, treated with acoustic energy, e.g., ultrasound.

In one example of the use of radiation as a pretreatment, half-gallonjuice cartons made of un-printed polycoated white Kraft board having abulk density of 20 lb/ft are used as a feedstock. Cartons are foldedflat and then fed into a sequence of three shredder-shearer trainsarranged in series with output from the first shearer fed as input tothe second shredder, and output from the second shearer fed as input tothe third shredder. The fibrous material produced by theshredder-shearer train can be sprayed with water and processed through apellet mill operating at room temperature. The densified pellets can beplaced in a glass ampoule, which is evacuated under high vacuum and thenback-filled with argon gas. The ampoule is sealed under argon.Alternatively, in another example, the ampoule is sealed under anatmosphere of air. The pellets in the ampoule are irradiated with gammaradiation for about 3 hours at a dose rate of about 1 Mrad per hour toprovide an irradiated material in which the cellulose has a lowermolecular weight than the starting material.

Additives to Enhance Molecular Weight Breakdown During Irradiation

In some embodiments, prior to irradiation, various materials, e.g.,solids or liquids, can be added to the biomass to enhance molecularweight reduction. In those instances in which a liquid is utilized, theliquid can be in contact with outer surfaces of the biomass and/or theliquid can be in interior portions of the biomass, e.g., infused intothe biomass.

For example, the material can be a neutral weak base, such as alanine,ammonia, ammonia/water mixture, e.g., 25 percent by weight ammonia inwater, water, methyl amine, dimethyl amine, trimethyl amine, pyridine,or a anionic base, such as a salt of acetic acid (e.g., sodium acetate),sodium carbonate, sodium bicarbonate or a salt of an ion of hydrogensulfide (e.g., sodium hydrosulfide).

Alternatively, the material can be a neutral weak acid, such as formicacid, acetic acid, trichloroacetic acid, water, hydrogen sulfide or acationic acid, such as an ammonium salt.

Quenching and Controlled Functionalization of Biomass

After treatment with one or more ionizing radiations, such as photonicradiation (e.g., X-rays or gamma-rays), e-beam radiation or particlesheavier than electrons that are positively or negatively charged (e.g.,protons or carbon ions), any of the carbohydrate-containing materials ormixtures described herein become ionized; that is, they include radicalsat levels that are detectable with an electron spin resonancespectrometer. The current practical limit of detection of the radicalsis about 10-14 spins at room temperature. After ionization, any biomassmaterial that has been ionized can be to reduce the level of radicals inthe ionized biomass, e.g., such that the radicals are no longerdetectable with the electron spin resonance spectrometer. For example,the radicals can be quenched by the application of a sufficient pressureto the biomass and/or by utilizing a fluid in contact with the ionizedbiomass, such as a gas or liquid, that reacts with (quenches) theradicals. The use of a gas or liquid to at least aid in the quenching ofthe radicals also allows the operator to control functionalization ofthe ionized biomass with a desired amount and kind of functional groups,such as carboxylic acid groups, enol groups, aldehyde groups, nitrogroups, nitrile groups, amino groups, alkyl amino groups, alkyl groups,chloroalkyl groups or chlorofluoroalkyl groups. In some instances, suchquenching can improve the stability of some of the ionized biomassmaterials. For example, quenching can improve the resistance of thebiomass to oxidation. Functionalization by quenching can also improvethe solubility of any biomass described herein, can improve its thermalstability, which can be important in the manufacture of composites andboards described herein, and can improve material utilization by variousmicroorganisms. For example, the functional groups imparted to thebiomass material by quenching can act as receptor sites for attachmentby microorganisms, e.g., to enhance cellulose hydrolysis by variousmicroorganisms.

FIG. 11B illustrates changing a molecular and/or a supramolecularstructure of a biomass feedstock by pretreating the biomass feedstockwith ionizing radiation, such as with electrons or ions of sufficientenergy to ionize the biomass feedstock, to provide a first level ofradicals. As shown in FIG. 11B, if the ionized biomass remains in theatmosphere, it will be oxidized, such as to an extent that carboxylicacid groups are generated by reacting with the atmospheric oxygen. Insome instances with some materials, such oxidation is desired because itcan aid in the further breakdown in molecular weight of thecarbohydrate-containing biomass, and the oxidation groups, e.g.,carboxylic acid groups can be helpful for solubility and microorganismutilization in some instances. However, since the radicals can “live”for some time after irradiation, e.g., longer than 1 day, 5 days, 30days, 3 months, 6 months or even longer than 1 year, material propertiescan continue to change over time, which in some instances, can beundesirable. Detecting radicals in irradiated samples by electron spinresonance spectroscopy and radical lifetimes in such samples isdiscussed in Bartolotta et al., Physics in Medicine and Biology, 46(2001), 461-471 and in Bartolotta et al., Radiation ProtectionDosimetry, Vol. 84, Nos. 1-4, pp. 293-296 (1999), which are attachedhereto as Appendix F and Appendix G, respectively. As shown in FIG. 11B,the ionized biomass can be quenched to functionalize and/or to stabilizethe ionized biomass. At any point, e.g., when the material is “alive”,“partially alive” or fully quenched, the pretreated biomass can beconverted into a product, e.g., a fuel, a food, or a composite.

In some embodiments, the quenching includes an application of pressureto the biomass, such as by mechanically deforming the biomass, e.g.,directly mechanically compressing the biomass in one, two, or threedimensions, or applying pressure to a fluid in which the biomass isimmersed, e.g., isostatic pressing. In such instances, the deformationof the material itself brings radicals, which are often trapped incrystalline domains, in sufficient proximity so that the radicals canrecombine, or react with another group. In some instances, the pressureis applied together with the application of heat, such as a sufficientquantity of heat to elevate the temperature of the biomass to above amelting point or softening point of a component of the biomass, such aslignin, cellulose or hemicellulose. Heat can improve molecular mobilityin the polymeric material, which can aid in the quenching of theradicals. When pressure is utilized to quench, the pressure can begreater than about 1000 psi, such as greater than about 1250 psi, 1450psi, 3625 psi, 5075 psi, 7250 psi, 10000 psi or even greater than 15000psi.

In some embodiments, quenching includes contacting the biomass with afluid, such as a liquid or gas, e.g., a gas capable of reacting with theradicals, such as acetylene or a mixture of acetylene in nitrogen,ethylene, chlorinated ethylenes or chlorofluoroethylenes, propylene ormixtures of these gases. In other particular embodiments, quenchingincludes contacting the biomass with a liquid, e.g., a liquid solublein, or at least capable of penetrating into the biomass and reactingwith the radicals, such as a diene, such as 1,5-cyclooctadiene. In somespecific embodiments, the quenching includes contacting the biomass withan antioxidant, such as Vitamin E. If desired, the biomass feedstock caninclude an antioxidant dispersed therein, and the quenching can comefrom contacting the antioxidant dispersed in the biomass feedstock withthe radicals.

Other methods for quenching are possible. For example, any method forquenching radicals in polymeric materials described in Muratoglu et al.,U.S. Patent Application Publication No. 2008/0067724 and Muratoglu etal., U.S. Pat. No. 7,166,650, which are attached as Appendix HandAppendix I, respectively, can be utilized for quenching any ionizedbiomass material described herein. Furthermore any quenching agent(described as a “sensitizing agent” in the above-noted Muratogludisclosures) and/or any antioxidant described in either Muratoglureference can be utilized to quench any ionized biomass material.

Functionalization can be enhanced by utilizing heavy charged ions, suchas any of the heavier ions described herein. For example, if it isdesired to enhance oxidation, charged oxygen ions can be utilized forthe irradiation. If nitrogen functional groups are desired, nitrogenions or ions that includes nitrogen can be utilized. Likewise, if sulfuror phosphorus groups are desired, sulfur or phosphorus ions can be usedin the irradiation.

In some embodiments, after quenching any of the quenched materialsdescribed herein can be further treated with one or more of radiation,such as ionizing or non-ionizing radiation, sonication, pyrolysis, andoxidation for additional molecular and/or supramolecular structurechange.

In particular embodiments, functionalized materials described herein aretreated with an acid, base, nucleophile or Lewis acid for additionalmolecular and/or supramolecular structure change, such as additionalmolecular weight breakdown. Examples of acids include organic acids,such as acetic acid and mineral acids, such as hydrochloric, sulfuricand/or nitric acid. Examples of bases include strong mineral bases, suchas a source of hydroxide ion, basic ions, such as fluoride ion, orweaker organic bases, such as amines. Even water and sodium bicarbonate,e.g., when dissolved in water, can effect molecular and/orsupramolecular structure change, such as additional molecular weightbreakdown.

Particle Beam Exposure in Fluids

In some cases, the cellulosic or lignocellulosic materials can beexposed to a particle beam in the presence of one or more additionalfluids (e.g., gases and/or liquids). Exposure of a material to aparticle beam in the presence of one or more additional fluids canincrease the efficiency of the treatment.

In some embodiments, the material is exposed to a particle beam in thepresence of a fluid such as air. Particles accelerated in any one ormore of the types of accelerators disclosed herein (or another type ofaccelerator) are coupled out of the accelerator via an output port(e.g., a thin membrane such as a metal foil), pass through a volume ofspace occupied by the fluid, and are then incident on the material. Inaddition to directly treating the material, some of the particlesgenerate additional chemical species by interacting with fluid particles(e.g., ions and/or radicals generated from various constituents of air,such as ozone and oxides of nitrogen). These generated chemical speciescan also interact with the material, and can act as initiators for avariety of different chemical bond-breaking reactions in the material.For example, any oxidant produced can oxidize the material, which canresult in molecular weight reduction.

In certain embodiments, additional fluids can be selectively introducedinto the path of a particle beam before the beam is incident on thematerial. As discussed above, reactions between the particles of thebeam and the particles of the introduced fluids can generate additionalchemical species, which react with the material and can assist infunctionalizing the material, and/or otherwise selectively alteringcertain properties of the material. The one or more additional fluidscan be directed into the path of the beam from a supply tube, forexample. The direction and flow rate of the fluid(s) that is/areintroduced can be selected according to a desired exposure rate and/ordirection to control the efficiency of the overall treatment, includingeffects that result from both particle-based treatment and effects thatare due to the interaction of dynamically generated species from theintroduced fluid with the material. In addition to air, exemplary fluidsthat can be introduced into the ion beam include oxygen, nitrogen, oneor more noble gases, one or more halogens, and hydrogen.

Irradiating Low Bulk Density Biomass Materials and Cooling IrradiatedBiomass

During treatment of biomass materials with ionizing radiation,especially at high dose rates, such as at rates greater than 0.15 Mradper second, e.g., 0.25 Mrad/s, 0.35 Mrad/s, 0.5 Mrad/s, 0.75 Mrad/s oreven greater than 1 Mrad/sec, biomass materials can retain significantquantities of heat so that the temperature of the biomass materialsbecomes elevated. While higher temperatures can, in some embodiments, beadvantageous, e.g., when a faster reaction rate is desired, it isadvantageous to control the heating of the biomass to retain controlover the chemical reactions initiated by the ionizing radiation, such ascross-linking, chain scission and/or grafting, e.g., to maintain processcontrol. Low bulk density materials, such as those having a bulk densityof less than about 0.4 g/cm³, e.g., less than about 0.35, 0.25 or lessabout 0.15 g/cm³, especially when combined with materials that have thincross-sections, such as fibers having small transverse dimensions, aregenerally easier to cool. In addition, photons and particles cangenerally penetrate further into and through materials having arelatively low bulk density, which can allow for the processing oflarger volumes of materials at higher rates, and can allow for the useof photons and particles that having lower energies, e.g., 0.25 MeV, 0.5MeV, 0.75 MeV or 1.0 MeV, which can reduce safety shieldingrequirements. For example, in one method of changing a molecular and/ora supramolecular structure of a biomass feedstock, the biomass ispretreated at a first temperature with ionizing radiation, such asphotons, electrons or ions (e.g., singularly or multiply charged cationsor anions), for a sufficient time and/or a sufficient dose to elevatethe biomass feedstock to a second temperature higher than the firsttemperature. The pretreated biomass is then cooled to a thirdtemperature below the second temperature. Finally, if desired, thecooled biomass can be treated one or more times with radiation, e.g.,with ionizing radiation. If desired, cooling can be applied to thebiomass after and/or during each radiation treatment.

The biomass feedstock can be physically prepared as discussed above,e.g., by reducing one or more dimensions of individual pieces of thebiomass feedstock so that the feedstock can be more efficientlyprocessed, e.g., more easily cooled and/or more easily penetrated by anionizing radiation.

In some implementations, the ionizing radiation is applied at a totaldose of less than 25 Mrad or less than 10 Mrad, such as less than 5 Mrador less than 2.5 Mrad, and at a rate of more than 0.25 Mrad per second,such as more than 0.5, 0.75 or greater than 1.0 Mrad/s, prior to coolingthe biomass.

The pretreating of the biomass feedstock with ionizing radiation can beperformed as the biomass feedstock is being pneumatically conveyed in afluid, such as a in a gas, e.g., nitrogen or air. To aid in molecularweight breakdown and/or functionalization of the materials, the gas canbe saturated with any swelling agent described herein and/or watervapor. For example, acidic water vapor can be utilized. To aid inmolecular weight breakdown, the water can be acidified with an organicacid, such as formic, or acetic acid, or a mineral acid, such assulfuric or hydrochloric acid.

The pretreating of the biomass feedstock with ionizing radiation can beperformed as the biomass feedstock falls under the influence of gravity.This procedure can effectively reduce the bulk density of the biomassfeedstock as it is being processed and can aid in the cooling of thebiomass feedstock. For example, the biomass can be conveyed from a firstbelt at a first height above the ground and then can be captured by asecond belt at a second level above the ground lower than the firstlevel. For example, in some embodiments, the trailing edge of the firstbelt and the leading edge of the second belt define a gap.Advantageously, the ionizing radiation, such as a beam of electrons,protons, or other ions, can be applied at the gap to prevent damage tothe biomass conveyance system.

Cooling of the biomass can include contacting the biomass with a fluid,such as a gas, at a temperature below the first or second temperature,such as gaseous nitrogen at or about 77 K. Even water, such as water ata temperature below nominal room temperature (e.g., 25 degrees Celsius)can be utilized.

Often advantageously, the biomass feedstock has internal fibers, andprior to irradiation with the ionizing radiation, the biomass feedstockhas been sheared to an extent that its internal fibers are substantiallyexposed. This shearing can provide a low bulk density material havingsmall cross-sectional dimensions, which can aid in the breakdown and/orfunctionalization of the biomass. For example, in some embodiments, thebiomass is or includes discrete fibers and/or particles having a maximumdimension of not more than about 0.5 mm, such as not more than about0.25 mm, not more than about 0.1 mm or not more than about 0.05 mm.

In some embodiments, the biomass feedstock to which the ionizingradiation is applied has a bulk density of less than about 0.35 g/cm³,such as less than about 0.3, 0.25, 0.20, or less than about 0.15 g/cm³during the application of the ionizing radiation. In such embodiments,the biomass feedstock can be cooled, and then ionizing radiation can beapplied to the cooled biomass. In some advantageous embodiments, thebiomass feedstock is or includes discrete fibers and/or particles havinga maximum dimension of not more than about 0.5 mm, such as not more thanabout 0.25 mm, not more than about 20.1 mm, not more than about 0.05 mm,or not more than about 0.025 mm.

Sonication

One or more sonication processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences. Sonication can reduce the molecular weight and/orcrystallinity of feedstock.

Referring again to FIG. 8, in one method, a first material 2 thatincludes cellulose having a first number average molecular weight(^(T)M_(N1)) is dispersed in a medium, such as water, and sonicatedand/or otherwise cavitated, to provide a second material 3 that includescellulose having a second number average molecular weight (^(T)M_(N2))lower than the first number average molecular weight. The secondmaterial (or the first and second material in certain embodiments) canbe combined with a microorganism (e.g., a bacterium or a yeast) that canutilize the second and/or first material to produce a fuels that is orincludes hydrogen, an alcohol, an organic acid, a hydrocarbon ormixtures of any of these.

Since the second material has cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable, and/or soluble in a solution containing themicroorganism, e.g., at a concentration of greater than 106microorganisms/mL. These properties make the second material 3 moresusceptible to chemical, enzymatic, and/or microbial attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Sonicationcan also sterilize the materials, but should not be used while themicroorganisms are supposed to be alive.

In some embodiments, the second number average molecular weight(^(T)M_(N2)) is lower than the first number average molecular weight(^(T)M_(N1)) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, 50 percent, 60 percent, or even more than about 75 percent.

In some instances, the second material has cellulose that has ascrystallinity (TC2) that is lower than the crystallinity (TC1) of thecellulose of the first material. For example, (TC2) can be lower than(TC1) by more than about 10 percent, e.g., 15, 20, 25, 30, 35, 40, oreven more than about 50 percent.

In some embodiments, the starting crystallinity index (prior tosonication) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after sonication is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in certain embodiments, e.g., after extensivesonication, it is possible to have a crystallinity index of lower than 5percent. In some embodiments, the material after sonication issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto sonication) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after sonication is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive sonication, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(^(T)O₂) that is higher than the level of oxidation (^(T)O₁) of thefirst material. A higher level of oxidation of the material can aid inits dispersibility, swellability and/or solubility, further enhancingthe materials susceptibility to chemical, enzymatic or microbial attack.In some embodiments, to increase the level of the oxidation of thesecond material relative to the first material, the sonication isperformed in an oxidizing medium, producing a second material that ismore oxidized than the first material. For example, the second materialcan have more hydroxyl groups, aldehyde groups, ketone groups, estergroups or carboxylic acid groups, which can increase its hydrophilicity.

In some embodiments, the sonication medium is an aqueous medium. Ifdesired, the medium can include an oxidant, such as a peroxide (e.g.,hydrogen peroxide), a dispersing agent and/or a buffer. Examples ofdispersing agents include ionic dispersing agents, e.g., sodium laurylsulfate, and non-ionic dispersing agents, e.g., poly(ethylene glycol).

In other embodiments, the sonication medium is non-aqueous. For example,the sonication can be performed in a hydrocarbon, e.g., toluene orheptane, an ether, e.g., diethyl ether or tetrahydrofuran, or even in aliquefied gas such as argon, xenon, or nitrogen.

Without wishing to be bound by any particular theory, it is believedthat sonication breaks bonds in the cellulose by creating bubbles in themedium containing the cellulose, which grow and then violently collapse.During the collapse of the bubble, which can take place in less than ananosecond, the implosive force raises the local temperature within thebubble to about 5100 K (even higher in some instance; see, e.g., Suslicket al., Nature 434, 52-55) and generates pressures of from a few hundredatmospheres to over 1000 atmospheres or more. It is these hightemperatures and pressures that break the bonds. In addition, withoutwishing to be bound by any particular theory, it is believed thatreduced crystallinity arises, at least in part, from the extremely highcooling rates during collapse of the bubbles, which can be greater thanabout 1011 K/second. The high cooling rates generally do not allow thecellulose to organize and crystallize, resulting in materials that havereduced crystallinity. Ultrasonic systems and sonochemistry arediscussed in, e.g., Olli et al., U.S. Pat. No. 5,766,764; Roberts, U.S.Pat. No. 5,828,156; Mason, Chemistry with Ultrasound, Elsevier, Oxford,(1990); Suslick (editor), Ultrasound: its Chemical, Physical andBiological Effects, VCH, Weinheim, (1988); Price, “Current Trends inSonochemistry” Royal Society of Chemistry, Cambridge, (1992); Suslick etal., Ann. Rev. Mater. Sci. 29, 295, (1999); Suslick et al., Nature 353,414 (1991); Hiller et al., Phys. Rev. Lett. 69, 1182 (1992); Barber etal., Nature, 352, 414 (1991); Suslick et al., J. Am. Chem. Soc., 108,5641 (1986); Tang et al., Chem. Comm., 2119 (2000); Wang et al.,Advanced Mater., 12, 1137 (2000); Landau et al., J. of Catalysis, 201,22 (2001); Perkas et al., Chem. Comm., 988 (2001); Nikitenko et al.,Angew. Chem. Inter. Ed. (December 2001); Shafi et al., J. Phys. Chem B103, 3358 (1999); Avivi et al., J. Amer. Chem. Soc. 121, 4196 (1999);and Avivi et al., J. Amer. Chem. Soc. 122, 4331 (2000).

Sonication Systems

FIG. 12 shows a general system in which a cellulosic material stream1210 is mixed with a water stream 1212 in a reservoir 1214 to form aprocess stream 1216. A first pump 1218 draws process stream 1216 fromreservoir 1214 and toward a flow cell 1224. Ultrasonic transducer 1226transmits ultrasonic energy into process stream 1216 as the processstream flows through flow cell 1224. A second pump 1230 draws processstream 1216 from flow cell 1224 and toward subsequent processing.

Reservoir 1214 includes a first intake 1232 and a second intake 1234 influid communication with a volume 1236. A conveyor (not shown) deliverscellulosic material stream 1210 to reservoir 1214 through first intake1232. Water stream 1212 enters reservoir 1214 through second intake1234. In some embodiments, water stream 1212 enters volume 1236 along atangent establishing a swirling flow within volume 1236. In certainembodiments, cellulosic material stream 1210 and water stream 1212 canbe introduced into volume 1236 along opposing axes to enhance mixingwithin the volume.

Valve 1238 controls the flow of water stream 1212 through second intake1232 to produce a desired ratio of cellulosic material to water (e.g.,approximately 10% cellulosic material, weight by volume). For example,2000 tons/day of cellulosic material can be combined with 1 million to1.5 million gallons/day, e.g., 1.25 million gallons/day, of water.

Mixing of cellulosic material and water in reservoir 1214 is controlledby the size of volume 1236 and the flow rates of cellulosic material andwater into the volume. In some embodiments, volume 1236 is sized tocreate a minimum mixing residence time for the cellulosic material andwater. For example, when 2000 tons/day of cellulosic material and 1.25million gallons/day of water are flowing through reservoir 1214, volume1236 can be about 32,000 gallons to produce a minimum mixing residencetime of about 15 minutes.

Reservoir 1214 includes a mixer 1240 in fluid communication with volume1236.

Mixer 1240 agitates the contents of volume 1236 to disperse cellulosicmaterial throughout the water in the volume. For example, mixer 1240 canbe a rotating vane disposed in reservoir 1214. In some embodiments,mixer 1240 disperses the cellulosic material substantially uniformlythroughout the water.

Reservoir 1214 further includes an exit 1242 in fluid communication withvolume 1236 and process stream 1216. The mixture of cellulosic materialand water in volume 1236 flows out of reservoir 1214 via exit 1242. Exit1242 is arranged near the bottom of reservoir 1214 to allow gravity topull the mixture of cellulosic material and water out of reservoir 1214and into process stream 1216.

First pump 1218 (e.g., any of several recessed impeller vortex pumpsmade by Essco Pumps & Controls, Los Angeles, Calif.) moves the contentsof process stream 1216 toward flow cell 1224. In some embodiments, firstpump 1218 agitates the contents of process stream 1216 such that themixture of cellulosic material and water is substantially uniform atinlet 1220 of flow cell 1224. For example, first pump 1218 agitatesprocess stream 1216 to create a turbulent flow that persists along theprocess stream between the first pump and inlet 1220 of flow cell 1224.

Flow cell 1224 includes a reactor volume 1244 in fluid communicationwith inlet 1220 and outlet 1222. In some embodiments, reactor volume1244 is a stainless steel tube capable of withstanding elevatedpressures (e.g., 10 bars). In addition or in the alternative, reactorvolume 1244 includes a rectangular cross section.

Flow cell 1224 further includes a heat exchanger 1246 in thermalcommunication with at least a portion of reactor volume 1244. Coolingfluid 1248 (e.g., water) flows into heat exchanger 1246 and absorbs heatgenerated when process stream 1216 is sonicated in reactor volume 1244.In some embodiments, the flow rate and/or the temperature of coolingfluid 1248 into heat exchanger 1246 is controlled to maintain anapproximately constant temperature in reactor volume 1244. In someembodiments, the temperature of reactor volume 1244 is maintained at 20to 50° C., e.g., 25, 30, 35, 40, or 45° C. Additionally oralternatively, heat transferred to cooling fluid 1248 from reactorvolume 1244 can be used in other parts of the overall process.

An adapter section 1226 creates fluid communication between reactorvolume 1244 and a booster 1250 coupled (e.g., mechanically coupled usinga flange) to ultrasonic transducer 1226. For example, adapter section1226 can include a flange and 0-ring assembly arranged to create a leaktight connection between reactor volume 1244 and booster 1250. In someembodiments, ultrasonic transducer 1226 is a high-powered ultrasonictransducer made by Hielscher Ultrasonics of Teltow, Germany.

In operation, a generator 1252 delivers electricity to ultrasonictransducer 1252. Ultrasonic transducer 1226 includes a piezoelectricelement that converts the electrical energy into sound in the ultrasonicrange. In some embodiments, the materials are sonicated using soundhaving a frequency of from about 16 kHz to about 110 kHz, e.g., fromabout 18 kHz to about 75 kHz or from about 20 kHz to about 40 kHz.(e.g., sound having a frequency of 20 kHz to 40 kHz). In some examples,sonication is performed at a frequency of between about 15 kHz and about25 kHz, such as between about 18 kHz and 22 kHz. In specificembodiments, sonicating can be performed utilizing a 1 KW or largerhorn, e.g., a 2, 3, 4, 5, or even a 10 KW horn.

The ultrasonic energy is then delivered to the working medium throughbooster 1248. The ultrasonic energy traveling through booster 1248 inreactor volume 1244 creates a series of compressions and rarefactions inprocess stream 1216 with an intensity sufficient to create cavitation inprocess stream 1216. Cavitation disaggregates the cellulosic materialdispersed in process stream 1216. Cavitation also produces free radicalsin the water of process stream 1216. These free radicals act to furtherbreak down the cellulosic material in process stream 1216.

In general, 5 to 4000 MJ/m3, e.g., 10, 25, 50, 100, 250, 500, 750, 1000,2000, or 3000 MJ/m3, of ultrasonic energy is applied to process stream16 flowing at a rate of about 0.2 m3/s (about 3200 gallons/min). Afterexposure to ultrasonic energy in reactor volume 1244, process stream1216 exits flow cell 1224 through outlet 1222. Second pump 1230 movesprocess stream 1216 to subsequent processing (e.g., any of severalrecessed impeller vortex pumps made by Essco Pumps & Controls, LosAngeles, Calif.).

While certain embodiments have been described, other embodiments arepossible. As an example, while process stream 1216 has been described asa single flow path, other arrangements are possible. In some embodimentsfor example, process stream 1216 includes multiple parallel flow paths(e.g., flowing at a rate of 10 gallon/min). In addition or in thealternative, the multiple parallel flow paths of process stream 1216flow into separate flow cells and are sonicated in parallel (e.g., usinga plurality of 16 kW ultrasonic transducers).

As another example, while a single ultrasonic transducer 1226 has beendescribed as being coupled to flow cell 1224, other arrangements arepossible. In some embodiments, a plurality of ultrasonic transducers1226 are arranged in flow cell 1224 (e.g., ten ultrasonic transducerscan be arranged in a flow cell 1224). In some embodiments, the soundwaves generated by each of the plurality of ultrasonic transducers 1226are timed (e.g., synchronized out of phase with one another) to enhancethe cavitation acting upon process stream 1216.

As another example, while a single flow cell 1224 has been described,other arrangements are possible. In some embodiments, second pump 1230moves process stream to a second flow cell where a second booster andultrasonic transducer further sonicate process stream 1216.

As still another example, while reactor volume 1244 has been describedas a closed volume, reactor volume 1244 is open to ambient conditions incertain embodiments. In such embodiments, sonication pretreatment can beperformed substantially simultaneously with other pretreatmenttechniques. For example, ultrasonic energy can be applied to processstream 1216 in reactor volume 1244 while electron beams aresimultaneously introduced into process stream 1216.

As another example, while a flow-through process has been described,other arrangements are possible. In some embodiments, sonication can beperformed in a batch process. For example, a volume can be filled with a10% (weight by volume) mixture of cellulosic material in water andexposed to sound with intensity from about 50 W/cm2 to about 600 W/cm2,e.g., from about 75 W/cm2 to about 300 W/cm2 or from about 95 W/cm2 toabout 200 W/cm2. Additionally or alternatively, the mixture in thevolume can be sonicated from about 1 hour to about 24 hours, e.g., fromabout 1.5 hours to about 12 hours, or from about 2 hours to about 10hours. In certain embodiments, the material is sonicated for apre-determined time, and then allowed to stand for a secondpre-determined time before sonicating again.

Referring now to FIG. 13, in some embodiments, two-electro-acoustictransducers are mechanically coupled to a single horn. As shown, a pairof piezoelectric transducers 60 and 62 is coupled to a slotted bar horn64 by respective intermediate coupling horns 70 and 72, the latter alsobeing known as booster horns. The mechanical vibrations provided by thetransducers, responsive to high frequency electrical energy appliedthereto, are transmitted to the respective coupling horns, which may beconstructed to provide a mechanical gain, such as a ratio of 1 to 1.2.The horns are provided with a respective mounting flange 74 and 76 forsupporting the transducer and horn assembly in a stationary housing.

The vibrations transmitted from the transducers through the coupling orbooster horns are coupled to the input surface 78 of the horn and aretransmitted through the horn to the oppositely disposed output surface80, which, during operation, is in forced engagement with a workpiece(not shown) to which the vibrations are applied.

The high frequency electrical energy provided by the power supply 82 isfed to each of the transducers, electrically connected in parallel, viaa balancing transformer 84 and a respective series connected capacitor86 and 90, one capacitor connected in series with the electricalconnection to each of the transducers. The balancing transformer isknown also as “balun” standing for “balancing unit.” The balancingtransformer includes a magnetic core 92 and a pair of identical windings94 and 96, also termed the primary winding and secondary winding,respectively.

In some embodiments, the transducers include commercially availablepiezoelectric transducers, such as Branson Ultrasonics Corporationmodels 105 or 502, each designed for operation at 20 kHz and a maximumpower rating of 3 kW. The energizing voltage for providing maximummotional excursion at the output surface of the transducer is 930 voltrms. The current flow through a transducer may vary between zero and 3.5ampere depending on the load impedance. At 930 volt rms the outputmotion is approximately 20 microns. The maximum difference in terminalvoltage for the same motional amplitude, therefore, can be 186 volt.Such a voltage difference can give rise to large circulating currentsflowing between the transducers. The balancing unit 430 assures abalanced condition by providing equal current flow through thetransducers, hence eliminating the possibility of circulating currents.The wire size of the windings must be selected for the full load currentnoted above and the maximum voltage appearing across a winding input is93 volt.

As an alternative to using ultrasonic energy, high-frequency,rotor-stator devices can be utilized. This type of device produceshigh-shear, microcavitation forces which can disintegrate biomass incontact with such forces. Two commercially available high-frequency,rotor-stator dispersion devices are the Supraton™ devices manufacturedby Krupp Industrietechnik GmbH and marketed by Don-Oliver DeutschlandGmbH of Connecticut, and the Dispax™ devices manufactured and marketedby Ika-Works, Inc. of Cincinnati, Ohio. Operation of such amicrocavitation device is discussed in Stuart, U.S. Pat. No. 5,370,999.

While ultrasonic transducer 1226 has been described as including one ormore piezoelectric active elements to create ultrasonic energy, otherarrangements are possible. In some embodiments, ultrasonic transducer1226 includes active elements made of other types of magnetostrictivematerials (e.g., ferrous metals). Design and operation of such ahigh-powered ultrasonic transducer is discussed in Hansen et al., U.S.Pat. No. 6,624,539. In some embodiments, ultrasonic energy istransferred to process stream 16 through an electro-hydraulic system.

While ultrasonic transducer 1226 has been described as using theelectromagnetic response of magnetorestrictive materials to produceultrasonic energy, other arrangements are possible. In some embodiments,acoustic energy in the form of an intense shock wave can be applieddirectly to process stream 16 using an underwater spark. In someembodiments, ultrasonic energy is transferred to process stream 16through a thermo-hydraulic system. For example, acoustic waves of highenergy density can be produced by applying power across an enclosedvolume of electrolyte, thereby heating the enclosed volume and producinga pressure rise that is subsequently transmitted through a soundpropagation medium (e.g., process stream 1216). Design and operation ofsuch a thermo-hydraulic transducer is discussed in Hartmann et al., U.S.Pat. No. 6,383,152.

Pyrolysis

One or more pyrolysis processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences.

Referring again to the general schematic in FIG. 8, a first material 2that includes cellulose having a first number average molecular weight(^(T)M_(N1)) is pyrolyzed, e.g., by heating the first material in a tubefurnace, to provide a second material 3 that includes cellulose having asecond number average molecular weight (^(T)M_(N2)) lower than the firstnumber average molecular weight. The second material (or the first andsecond material in certain embodiments) is/are combined with amicroorganism (e.g., a bacterium or a yeast) that can utilize the secondand/or first material to produce a fuels that is or includes hydrogen,an alcohol (e.g., ethanol or butanol, such as n-, sec or t-butanol), anorganic acid, a hydrocarbon or mixtures of any of these.

Since the second material has cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable and/or soluble in a solution containing themicroorganism, e.g., at a concentration of greater than 106microorganisms/mL. These properties make the second material 3 moresusceptible to chemical, enzymatic and/or microbial attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Pyrolysiscan also sterilize the first and second materials.

In some embodiments, the second number average molecular weight (TMN2)is lower than the first number average molecular weight (TMN1) by morethan about 10 percent, e.g., 15, 20, 25, 30, 35, 40, 50 percent, 60percent, or even more than about 75 percent.

In some instances, the second material has cellulose that has ascrystallinity (TC2) that is lower than the crystallinity (TC1) of thecellulose of the first material. For example, (TC2) can be lower than(TC1) by more than about 10 percent, e.g., 15, 20, 25, 30, 35, 40, oreven more than about 50 percent.

In some embodiments, the starting crystallinity (prior to pyrolysis) isfrom about 40 to about 87.5 percent, e.g., from about 50 to about 75percent or from about 60 to about 70 percent, and the crystallinityindex after pyrolysis is from about 10 to about 50 percent, e.g., fromabout 15 to about 45 percent or from about 20 to about 40 percent.However, in certain embodiments, e.g., after extensive pyrolysis, it ispossible to have a

crystallinity index of lower than 5 percent. In some embodiments, thematerial after pyrolysis is substantially amorphous.

In some embodiments, the starting number average molecular weight (priorto pyrolysis) is from about 200,000 to about 3,200,000, e.g., from about250,000 to about 1,000,000 or from about 250,000 to about 700,000, andthe number average molecular weight after pyrolysis is from about 50,000to about 200,000, e.g., from about 60,000 to about 150,000 or from about70,000 to about 125,000. However, in some embodiments, e.g., afterextensive pyrolysis, it is possible to have a number average molecularweight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second material can have a level of oxidation(TO2) that is higher than the level of oxidation (TO1) of the firstmaterial. A higher level of oxidation of the material can aid in itsdispersibility, swellability and/or solubility, further enhancing thematerials susceptibility to chemical, enzymatic or microbial attack. Insome embodiments, to increase the level of the oxidation of the secondmaterial relative to the first material, the pyrolysis is performed inan oxidizing environment, producing a second material that is moreoxidized than the first material. For example, the second material canhave more hydroxyl groups, aldehyde groups, ketone groups, ester groupsor carboxylic acid groups, which can increase its hydrophilicity.

In some embodiments, the pyrolysis of the materials is continuous. Inother embodiments, the material is pyrolyzed for a pre-determined time,and then allowed to cool for a second pre-determined time beforepyrolyzing again.

Pyrolysis Systems

FIG. 14 shows a process flow diagram 6000 that includes various steps ina pyrolytic feedstock pretreatment system. In first step 6010, a supplyof dry feedstock is received from a feed source.

As described above, the dry feedstock from the feed source may bepre-processed prior to delivery to the pyrolysis chamber. For example,if the feedstock is derived from plant sources, certain portions of theplant material may be removed prior to collection of the plant materialand/or before the plant material is delivered by the feedstock transportdevice. Alternatively, or in addition, the biomass feedstock can besubjected to mechanical processing 6020 (e.g., to reduce the averagelength of fibers in the feedstock) prior to delivery to the pyrolysischamber.

Following mechanical processing, the feedstock undergoes a moistureadjustment step 6030. The nature of the moisture adjustment step dependsupon the moisture content of the mechanically processed feedstock.Typically, pyrolysis of feedstock occurs most efficiently when themoisture content of the feedstock is between about 10% and about 30%(e.g., between 15% and 25%) by weight of the feedstock. If the moisturecontent of the feedstock is larger than about 40% by weight, the extrathermal load presented by the water content of the feedstock increasesthe energy consumption of subsequent pyrolysis steps.

In some embodiments, if the feedstock has a moisture content which islarger than about 30% by weight, drier feedstock material 6220 which hasa low moisture content can be blended in, creating a feedstock mixturein step 6030 with an average moisture content that is within the limitsdiscussed above. In certain embodiments, feedstock with a high moisturecontent can simply be dried by dispersing the feedstock material on amoving conveyor that cycles the feedstock through an in-line heatingunit. The heating unit evaporates a portion of the water present in thefeedstock.

In some embodiments, if the feedstock from step 6020 has a moisturecontent which is too low (e.g., lower than about 10% by weight), themechanically processed feedstock can be combined with wetter feedstockmaterial 6230 with a higher moisture content, such as sewage sludge.Alternatively, or in addition, water 6240 can be added to the dryfeedstock from step 6020 to increase its moisture content.

In step 6040, the feedstock—now with its moisture content adjusted tofall within suitable limits—can be preheated in an optional preheatingstep 6040. Preheating step 6040 can be used to increase the temperatureof the feedstock to between 75° C. and 150° C. in preparation forsubsequent pyrolysis of the feedstock. Depending upon the nature of thefeedstock and the particular design of the pyrolysis chamber, preheatingthe feedstock can ensure that heat distribution within the feedstockremains more uniform during pyrolysis, and can reduce the thermal loadon the pyrolysis chamber.

The feedstock is then transported to a pyrolysis chamber to undergopyrolysis in step 6050. In some embodiments, transport of the feedstockis assisted by adding one or more pressurized gases 6210 to thefeedstock stream. The gases create a pressure gradient in a feedstocktransport conduit, propelling the feedstock into the pyrolysis chamber(and even through the pyrolysis chamber). In certain embodiments,transport of the feedstock occurs mechanically; that is, a transportsystem that includes a conveyor such as an auger transports thefeedstock to the pyrolysis chamber.

Other gases 6210 can also be added to the feedstock prior to thepyrolysis chamber. In some embodiments, for example, one or morecatalyst gases can be added to the feedstock to assist decomposition ofthe feedstock during pyrolysis. In certain embodiments, one or morescavenging agents can be added to the feedstock to trap volatilematerials released during pyrolysis. For example, various sulfur-basedcompounds such as sulfides can be liberated during pyrolysis, and anagent such as hydrogen gas can be added to the feedstock to causedesulfurization of the pyrolysis products. Hydrogen combines withsulfides to form hydrogen sulfide gas, which can be removed from thepyrolyzed feedstock.

Pyrolysis of the feedstock within the chamber can include heating thefeedstock to relatively high temperatures to cause partial decompositionof the feedstock. Typically, the feedstock is heated to a temperature ina range from 150° C. to 1100° C. The temperature to which the feedstockis heated depends upon a number of factors, including the composition ofthe feedstock, the feedstock average particle size, the moisturecontent, and the desired pyrolysis products. For many types of biomassfeedstock, for example, pyrolysis temperatures between 300° C. and 550°C. are used.

The residence time of the feedstock within the pyrolysis chambergenerally depends upon a number of factors, including the pyrolysistemperature, the composition of the feedstock, the feedstock averageparticle size, the moisture content, and the desired pyrolysis products.In some embodiments, feedstock materials are pyrolyzed at a temperaturejust above the decomposition temperature for the material in an inertatmosphere, e.g., from about 2° C. above to about 10° C. above thedecomposition temperature or from about 3° C. above to about 7° C. abovethe decomposition temperature. In such embodiments, the material isgenerally kept at this temperature for greater than 0.5 hours, e.g.,greater than 1.0 hour or greater than about 2.0 hours. In otherembodiments, the materials are pyrolyzed at a temperature well above thedecomposition temperature for the material in an inert atmosphere, e.g.,from about 75° C. above to about 175° C. above the decompositiontemperature or from about 85° C. above to about 150° C. above thedecomposition temperature. In such embodiments, the material isgenerally kept at this temperature for less than 0.5 hour, e.g., less 20minutes, less than 10 minutes, less than 5 minutes or less than 2minutes. In still other embodiments, the materials are pyrolyzed at anextreme temperature, e.g., from about 200° C. above to about 500° C.above the decomposition temperature of the material in an inertenvironment or from about 250° C. above to about 400° C. above thedecomposition temperature. In such embodiments, the material isgenerally kept at this temperature for less than 1 minute, e.g., lessthan 30 seconds, 15 seconds, 10 seconds, 5 seconds, 1 second or lessthan 500 ms. Such embodiments are typically referred to as flashpyrolysis.

In some embodiments, the feedstock is heated relatively rapidly to theselected pyrolysis temperature within the chamber. For example, thechamber can be designed to heat the feedstock at a rate of between 500°C. and 11,000° C., for example from 500° C. to 1000° C.

A turbulent flow of feedstock material within the pyrolysis chamber isusually advantageous, as it ensures relatively efficient heat transferto the feedstock material from the heating sub-system. Turbulent flowcan be achieved, for example, by blowing the feedstock material throughthe chamber using one or more injected carrier gases 6210. In general,the carrier gases are relatively inert towards the feedstock material,even at the high temperatures in the pyrolysis chamber. Exemplarycarrier gases include, for example, nitrogen, argon, methane, carbonmonoxide, and carbon dioxide. Alternatively, or in addition, mechanicaltransport systems such as augers can transport and circulate thefeedstock within the pyrolysis chamber to create a turbulent feedstockflow.

In some embodiments, pyrolysis of the feedstock occurs substantially inthe absence of oxygen and other reactive gases. Oxygen can be removedfrom the pyrolysis chamber by periodic purging of the chamber with highpressure nitrogen (e.g., at nitrogen pressures of 2 bar or more).Following purging of the chamber, a gas mixture present in the pyrolysischamber (e.g., during pyrolysis of the feedstock) can include less than4 mole % oxygen (e.g., less than 1 mole % oxygen, and even less than 0.5mole % oxygen). The absence of oxygen ensures that ignition of thefeedstock does not occur at the elevated pyrolysis temperatures.

In certain embodiments, relatively small amounts of oxygen can beintroduced into the feedstock and are present during pyrolysis. Thistechnique is referred to as oxidative pyrolysis. Typically, oxidativepyrolysis occurs in multiple heating stages. For example, in a firstheating stage, the feedstock is heated in the presence of oxygen tocause partial oxidation of the feedstock. This stage consumes theavailable oxygen in the pyrolysis chamber. Then, in subsequent heatingstages, the feedstock temperature is further elevated. With all of theoxygen in the chamber consumed, however, feedstock combustion does notoccur, and combustion-free pyrolytic decomposition of the feedstock(e.g., to generate hydrocarbon products) occurs. In general, the processof heating feedstock in the pyrolysis chamber to initiate decompositionis endothermic. However, in oxidative pyrolysis, formation of carbondioxide by oxidation of the feedstock is an exothermic process. The heatreleased from carbon dioxide formation can assist further pyrolysisheating stages, thereby lessening the thermal load presented by thefeedstock.

In some embodiments, pyrolysis occurs in an inert environment, such aswhile feedstock materials are bathed in argon or nitrogen gas. Incertain embodiments, pyrolysis can occur in an oxidizing environment,such as in air or argon enriched in air. In some embodiments, pyrolysiscan take place in a reducing environment, such as while feedstockmaterials are bathed in hydrogen gas. To aid pyrolysis, various chemicalagents, such as oxidants, reductants, acids or bases can be added to thematerial prior to or during pyrolysis. For example, sulfuric acid can beadded, or a peroxide (e.g., benzoyl peroxide) can be added.

As discussed above, a variety of different processing conditions can beused, depending upon factors such as the feedstock composition and thedesired pyrolysis products. For example, for cellulose-containingfeedstock material, relatively mild pyrolysis conditions can beemployed, including flash pyrolysis temperatures between 375° C. and450° C., and residence times of less than 1 second. As another example,for organic solid waste material such as sewage sludge, flash pyrolysistemperatures between 500° C. and 650° C. are typically used, withresidence times of between 0.5 and 3 seconds. In general, many of thepyrolysis process parameters, including residence time, pyrolysistemperature, feedstock turbulence, moisture content, feedstockcomposition, pyrolysis product composition, and additive gas compositioncan be regulated automatically by a system of regulators and anautomated control system.

Following pyrolysis step 6050, the pyrolysis products undergo aquenching step 6250 to reduce the temperature of the products prior tofurther processing. Typically, quenching step 6250 includes spraying thepyrolysis products with streams of cooling water 6260. The cooling wateralso forms a slurry that includes solid, undissolved product materialand various dissolved products. Also present in the product stream is amixture that includes various gases, including product gases, carriergases, and other types of process gases.

The product stream is transported via in-line piping to a gas separatorthat performs a gas separation step 6060, in which product gases andother gases are separated from the slurry formed by quenching thepyrolysis products. The separated gas mixture is optionally directed toa blower 6130, which increases the gas pressure by blowing air into themixture. The gas mixture can be subjected to a filtration step 6140, inwhich the gas mixture passes through one or more filters (e.g.,activated charcoal filters) to remove particulates and other impurities.In a subsequent step 6150, the filtered gas can be compressed and storedfor further use. Alternatively, the filtered gas can be subjected tofurther processing steps 6160. For example, in some embodiments, thefiltered gas can be condensed to separate different gaseous compoundswithin the gas mixture. The different compounds can include, forexample, various hydrocarbon products (e.g., alcohols, alkanes, alkenes,alkynes, ethers) produced during pyrolysis. In certain embodiments, thefiltered gas containing a mixture of hydrocarbon components can becombined with steam gas 6170 (e.g., a mixture of water vapor and oxygen)and subjected to a cracking process to reduce molecular weights of thehydrocarbon components.

In some embodiments, the pyrolysis chamber includes heat sources thatburn hydrocarbon gases such as methane, propane, and/or butane to heatthe feedstock. A portion 6270 of the separated gases can be recirculatedinto the pyrolysis chamber for combustion, to generate process heat tosustain the pyrolysis process.

In certain embodiments, the pyrolysis chamber can receive process heatthat can be used to increase the temperature of feedstock materials. Forexample, irradiating feedstock with radiation (e.g., gamma radiation,electron beam radiation, or other types of radiation) can heat thefeedstock materials to relatively high temperatures. The heatedfeedstock materials can be cooled by a heat exchange system that removessome of the excess heat from the irradiated feedstock. The heat exchangesystem can be configured to transport some of the heat energy to thepyrolysis chamber to heat (or pre-heat) feedstock material, therebyreducing energy cost for the pyrolysis process.

The slurry containing liquid and solid pyrolysis products can undergo anoptional de-watering step 6070, in which excess water can be removedfrom the slurry via processes such as mechanical pressing andevaporation. The excess water 6280 can be filtered and then recirculatedfor further use in quenching the pyrolysis decomposition products instep 6250.

The de-watered slurry then undergoes a mechanical separation step 6080,in which solid product material 6110 is separated from liquid productmaterial 6090 by a series of increasingly fine filters. In step 6100,the liquid product material 6090 can then be condensed (e.g., viaevaporation) to remove waste water 6190, and purified by processes suchas extraction. Extraction can include the addition of one or moreorganic solvents 6180, for example, to separate products such as oilsfrom products such as alcohols. Suitable organic solvents include, forexample, various hydrocarbons and halo-hydrocarbons. The purified liquidproducts 6200 can then be subjected to further processing steps. Wastewater 6190 can be filtered if necessary, and recirculated for furtheruse in quenching the pyrolysis decomposition products in step 6250.

After separation in step 6080, the solid product material 6110 isoptionally subjected to a drying step 6120 that can include evaporationof water. Solid material 6110 can then be stored for later use, orsubjected to further processing steps, as appropriate.

The pyrolysis process parameters discussed above are exemplary. Ingeneral, values of these parameters can vary widely according to thenature of the feedstock and the desired products. Moreover, a widevariety of different pyrolysis techniques, including using heat sourcessuch as hydrocarbon flames and/or furnaces, infrared lasers, microwaveheaters, induction heaters, resistive heaters, and other heating devicesand configurations can be used.

A wide variety of different pyrolysis chambers can be used to decomposethe feedstock. In some embodiments, for example, pyrolyzing feedstockcan include heating the material using a resistive heating member, suchas a metal filament or metal ribbon. The heating can occur by directcontact between the resistive heating member and the material.

In certain embodiments, pyrolyzing can include heating the material byinduction, such as by using a Curie-Point pyrolyzer. In someembodiments, pyrolyzing can include heating the material by theapplication of radiation, such as infrared radiation. The radiation canbe generated by a laser, such as an infrared laser.

In certain embodiments, pyrolyzing can include heating the material witha convective heat. The convective heat can be generated by a flowingstream of heated gas. The heated gas can be maintained at a temperatureof less than about 1200° C., such as less than 1000° C., less than 750°C., less than 600° C., less than 400° C. or even less than 300° C. Theheated gas can be maintained at a temperature of greater than about 250°C. The convective heat can be generated by a hot body surrounding thefirst material, such as in a furnace.

In some embodiments, pyrolyzing can include heating the material withsteam at a temperature above about 250° C.

An embodiment of a pyrolysis chamber is shown in FIG. 15. Chamber 6500includes an insulated chamber wall 6510 with a vent 6600 for exhaustgases, a plurality of burners 6520 that generate heat for the pyrolysisprocess, a transport duct 6530 for transporting the feedstock throughchamber 6500, augers 6590 for moving the feedstock through duct 6530 ina turbulent flow, and a quenching system 6540 that includes an auger6610 for moving the pyrolysis products, water jets 6550 for spraying thepyrolysis products with cooling water, and a gas separator forseparating gaseous products 6580 from a slurry 6570 containing solid andliquid products.

Another embodiment of a pyrolysis chamber is shown in FIG. 16. Chamber6700 includes an insulated chamber wall 6710, a feedstock supply duct6720, a sloped inner chamber wall 6730, burners 6740 that generate heatfor the pyrolysis process, a vent 6750 for exhaust gases, and a gasseparator 6760 for separating gaseous products 6770 from liquid andsolid products 6780. Chamber 6700 is configured to rotate in thedirection shown by arrow 6790 to ensure adequate mixing and turbulentflow of the feedstock within the chamber.

A further embodiment of a pyrolysis chamber is shown in FIG. 17.Filament pyrolyzer 1712 includes a sample holder 1713 with resistiveheating element 1714 in the form of a wire winding through the openspace defined by the sample holder 1713. Optionally, the heated elementcan be spun about axis 1715 (as indicated by arrow 1716) to tumble thematerial that includes the cellulosic material in sample holder 1713.The space 1718 defined by enclosure 1719 is maintained at a temperatureabove room temperature, e.g., 200 to 250° C. In a typical usage, acarrier gas, e.g., an inert gas, or an oxidizing or reducing gas,traverses through the sample holder 1713 while the resistive heatingelement is rotated and heated to a desired temperature, e.g., 325° C.After an appropriate time, e.g., 5 to 10 minutes, the pyrolyzed materialis emptied from the sample holder. The system shown in FIG. 17 can bescaled and made continuous. For example, rather than a wire as theheating member, the heating member can be an auger screw. Material cancontinuously fall into the sample holder, striking a heated screw thatpyrolizes the material. At the same time, the screw can push thepyrolyzed material out of the sample holder to allow for the entry offresh, unpyrolyzed material.

Another embodiment of a pyrolysis chamber is shown in FIG. 18, whichfeatures a Curie-Point pyrolyzer 1820 that includes a sample chamber1821 housing a ferromagnetic foil 1822. Surrounding the sample chamber1821 is an RF coil 1823. The space 1824 defined by enclosure 1825 ismaintained at a temperature above room temperature, e.g., 200 to 250° C.In a typical usage, a carrier gas traverses through the sample chamber1821 while the foil 1822 is inductively heated by an applied RF field topyrolize the material at a desired temperature.

Yet another embodiment of a pyrolysis chamber is shown in FIG. 19.Furnace pyrolyzer 130 includes a movable sample holder 131 and a furnace132. In a typical usage, the sample is lowered (as indicated by arrow137) into a hot zone 135 of furnace 132, while a carrier gas fills thehousing 136 and traverses through the sample holder 131. The sample isheated to the desired temperature for a desired time to provide apyrolyzed product. The pyrolyzed product is removed from the pyrolyzerby raising the sample holder (as indicated by arrow 134).

In certain embodiments, as shown in FIG. 20, a cellulosic target 140 canbe pyrolyzed by treating the target, which is housed in a vacuum chamber141, with laser

light, e.g., light having a wavelength of from about 225 nm to about1500 nm. For example, the target can be ablated at 266 nm, using thefourth harmonic of an Nd-YAG laser (Spectra Physics, GCR170, San Jose,Calif.). The optical configuration shown allows the nearly monochromaticlight 143 generated by the laser 142 to be directed using mirrors 144and 145 onto the target after passing through a lens 146 in the vacuumchamber 141. Typically, the pressure in the vacuum chamber is maintainedat less than about 10-6 mm Hg. In some embodiments, infrared radiationis used, e.g., 1.06 micron radiation from an Nd-YAG laser. In suchembodiments, an infrared sensitive dye can be combined with thecellulosic material to produce a cellulosic target. The infrared dye canenhance the heating of the cellulosic material. Laser ablation isdescribed by Blanchet-Fincher et al. in U.S. Pat. No. 5,942,649.

Referring to FIG. 21, in some embodiments, a cellulosic material can beflash pyrolyzed by coating a tungsten filament 150, such as a 5 to 25mil tungsten filament, with the desired cellulosic material while thematerial is housed in a vacuum chamber 151. To affect pyrolysis, currentis passed through the filament, which causes a rapid heating of thefilament for a desired time. Typically, the heating is continued forseconds before allowing the filament to cool. In some embodiments, theheating is performed a number of times to effect the desired amount ofpyrolysis.

In certain embodiments, carbohydrate-containing biomass material can beheated in an absence of oxygen in a fluidized bed reactor. If desired,the carbohydrate containing biomass can have relatively thincross-sections, and can include any of the fibrous materials describedherein, for efficient heat transfer. The material can be heated bythermal transfer from a hot metal or ceramic, such as glass beads orsand in the reactor, and the resulting pyrolysis liquid or oil can betransported to a central refinery for making combustible fuels or otheruseful products.

Oxidation

One or more oxidative processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences.

Referring again to FIG. 8, a first material 2 that includes cellulosehaving a first number average molecular weight (^(T)M_(N1)) and having afirst oxygen content (^(T)O₁) is oxidized, e.g., by heating the firstmaterial in a tube furnace in stream of air or oxygen-enriched air, toprovide a second material 3 that includes cellulose having a secondnumber average molecular weight (^(T)M_(N2)) and having a second oxygencontent (^(T)O₂) higher than the first oxygen content (^(T)O₁). Thesecond material (or the first and second material in certainembodiments) can be, e.g., combined with a resin, such as a moltenthermoplastic resin or a microorganism, to provide a composite 4 havingdesirable mechanical properties, or a fuel 5.

Such materials can also be combined with a solid and/or a liquid. Forexample, the liquid can be in the form of a solution and the solid canbe particulate in form. The liquid and/or solid can include amicroorganism, e.g., a bacterium, and/or an enzyme. For example, thebacterium and/or enzyme can work on the cellulosic or lignocellulosicmaterial to produce a fuel, such as ethanol, or a coproduct, such as aprotein. Fuels and coproducts are described in FIBROUS MATERIALS ANDCOMPOSITES,” U.S. Ser. No. 11/453,951, filed Jun. 15, 2006. The entirecontents of each of the foregoing applications are incorporated hereinby reference.

In some embodiments, the second number average molecular weight is notmore 97 percent lower than the first number average molecular weight,e.g., not more than 95 percent, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45,40, 30, 20, 12.5, 10.0, 7.5, 5.0, 4.0, 3.0, 2.5, 2.0 or not more than1.0 percent lower than the first number average molecular weight. Theamount of reduction of molecular weight will depend upon theapplication.

In some embodiments in which the materials are used to make a fuel or acoproduct, the starting number average molecular weight (prior tooxidation) is from about 200,000 to about 3,200,000, e.g., from about250,000 to about 1,000,000 or from about 250,000 to about 700,000, andthe number average molecular weight after oxidation is from about 50,000to about 200,000, e.g., from about 60,000 to about 150,000 or from about70,000 to about 125,000. However, in some embodiments, e.g., afterextensive oxidation, it is possible to have a number average molecularweight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second oxygen content is at least about fivepercent higher than the first oxygen content, e.g., 7.5 percent higher,10.0 percent higher, 12.5 percent higher, 15.0 percent higher or 17.5percent higher. In some preferred embodiments, the second oxygen contentis at least about 20.0 percent higher than the oxygen content of thefirst material. Oxygen content is measured by elemental analysis bypyrolyzing a sample in a furnace operating 130° C. or higher. A suitableelemental analyzer is the LECO CHNS-932 analyzer with a VTF-900 hightemperature pyrolysis furnace

In some embodiments, oxidation of first material 200 does not result ina substantial change in the crystallinity of the cellulose. However, insome instances, e.g., after extreme oxidation, the second material hascellulose that has as crystallinity (^(T)C₂) that is lower than thecrystallinity (^(T)C₁) of the cellulose of the first material. Forexample, (^(T)C₂) can be lower than (^(T)C₁) by more than about 5percent, e.g., 10, 15, 20, or even 25 percent. This can be desirable toenhance solubility of the materials in a liquid, such as a liquid thatincludes a bacterium and/or an enzyme.

In some embodiments, the starting crystallinity index (prior tooxidation) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after oxidation is from about 30 to about 75.0percent, e.g., from about 35.0 to about 70.0 percent or from about 37.5to about 65.0 percent. However, in certain embodiments, e.g., afterextensive oxidation, it is possible to have a crystallinity index oflower than 5 percent. In some embodiments, the material after oxidationis substantially amorphous.

Without wishing to be bound by any particular theory, it is believedthat oxidation increases the number of hydrogen-bonding groups on thecellulose, such as hydroxyl groups, aldehyde groups, ketone groupscarboxylic acid groups or anhydride groups, which can increase itsdispersibility and/or its solubility (e.g., in a liquid). To furtherimprove dispersibility in a resin, the resin can include a componentthat includes hydrogen-bonding groups, such as one or more anhydridegroups, carboxylic acid groups, hydroxyl groups, amide groups, aminegroups or mixtures of any of these groups. In some preferredembodiments, the component includes a polymer copolymerized with and/orgrafted with maleic anhydride. Such materials are available from Dupontunder the tradename FUSABOND®.

Generally, oxidation of first material 200 occurs in an oxidizingenvironment. For example, the oxidation can be effected or aided bypyrolysis in an oxidizing environment, such as in air or argon enrichedin air. To aid in the oxidation, various chemical agents, such asoxidants, acids or bases can be added to the material prior to or duringoxidation. For example, a peroxide (e.g., benzoyl peroxide) can be addedprior to oxidation.

Oxidation Systems

FIG. 22 shows a process flow diagram 5000 that includes various steps inan oxidative feedstock pretreatment system. In first step 5010, a supplyof dry feedstock is received from a feed source. The feed source caninclude, for example, a storage bed or container that is connected to anin-line oxidation reactor via a conveyor belt or another feedstocktransport device.

As described above, the dry feedstock from the feed source may bepre-processed prior to delivery to the oxidation reactor. For example,if the feedstock is derived from plant sources, certain portions of theplant material may be removed prior to collection of the plant materialand/or before the plant material is delivered by the feedstock transportdevice. Alternatively, or in addition, the biomass feedstock can besubjected to mechanical processing (e.g., to reduce the average lengthof fibers in the feedstock) prior to delivery to the oxidation reactor.

Following mechanical processing 5020, feedstock 5030 is transported to amixing system which introduces water 5150 into the feedstock in amechanical mixing process. Combining water with the processed feedstockin mixing step 5040 creates an aqueous feedstock slurry 5050, which canthen be treated with one or more oxidizing agents.

Typically, one liter of water is added to the mixture for every 0.02 kgto 1.0 kg of dry feedstock. The ratio of feedstock to water in themixture depends upon the source of the feedstock and the specificoxidizing agents used further downstream in the overall process. Forexample, in typical industrial processing sequences for lignocellulosicbiomass, aqueous feedstock slurry 5050 includes from about 0.5 kg toabout 1.0 kg of dry biomass per liter of water.

In some embodiments, one or more fiber-protecting additives 5170 canalso be added to the feedstock slurry in feedstock mixing step 5040.Fiber-protecting additives help to reduce degradation of certain typesof biomass fibers (e.g., cellulose fibers) during oxidation of thefeedstock. Fiber-protecting additives can be used, for example, if adesired product from processing a lignocellulosic feedstock includescellulose fibers. Exemplary fiber-protecting additives include magnesiumcompounds such as magnesium hydroxide. Concentrations offiber-protecting additives in feedstock slurry 5050 can be from 0.1% to0.4% of the dry weight of the biomass feedstock, for example.

In certain embodiments, aqueous feedstock slurry 5050 can be subjectedto an optional extraction 5180 with an organic solvent to removewater-insoluble substances from the slurry. For example, extraction ofslurry 5050 with one or more organic solvents yields a purified slurryand an organic waste stream 5210 that includes water-insoluble materialssuch as fats, oils, and other non-polar, hydrocarbon-based substances.Suitable solvents for performing extraction of slurry 5050 includevarious alcohols, hydrocarbons, and halo-hydrocarbons, for example.

In some embodiments, aqueous feedstock slurry 5050 can be subjected toan optional thermal treatment 5190 to further prepare the feedstock foroxidation. An example of a thermal treatment includes heating thefeedstock slurry in the presence of pressurized steam. In fibrousbiomass feedstock, the pressurized steam swells the fibers, exposing alarger fraction of fiber surfaces to the aqueous solvent and tooxidizing agents that are introduced in subsequent processing steps.

In certain embodiments, aqueous feedstock slurry 5050 can be subjectedto an optional treatment with basic agents 5200. Treatment with one ormore basic agents can help to separate lignin from cellulose inlignocellulosic biomass feedstock, thereby improving subsequentoxidation of the feedstock. Exemplary basic agents include alkali andalkaline earth hydroxides such as sodium hydroxide, potassium hydroxide,and calcium hydroxide. In general, a variety of basic agents can beused, typically in concentrations from about 0.01% to about 0.5% of thedry weight of the feedstock.

Aqueous feedstock slurry 5050 is transported (e.g., by an in-line pipingsystem) to a chamber, which can be an oxidation preprocessing chamber oran oxidation reactor. In oxidation preprocessing step 5060, one or moreoxidizing agents 5160 are added to feedstock slurry 5050 to form anoxidizing medium. In some embodiments, for example, oxidizing agents5160 can include hydrogen peroxide. Hydrogen peroxide can be added toslurry 5050 as an aqueous solution, and in proportions ranging from 3%to between 30% and 35% by weight of slurry 5050. Hydrogen peroxide has anumber of advantages as an oxidizing agent. For example, aqueoushydrogen peroxide solution is relatively inexpensive, is relativelychemically stable, and is not particularly hazardous relative to otheroxidizing agents (and therefore does not require burdensome handlingprocedures and expensive safety equipment). Moreover, hydrogen peroxidedecomposes to form water during oxidation of feedstock, so that wastestream cleanup is relatively straightforward and inexpensive.

In certain embodiments, oxidizing agents 5160 can include oxygen (e.g.,oxygen gas) either alone, or in combination with hydrogen peroxide.Oxygen gas can be bubbled into slurry 5050 in proportions ranging from0.5% to 10% by weight of slurry 5050. Alternatively, or in addition,oxygen gas can also be introduced into a gaseous phase in equilibriumwith slurry 5050 (e.g., a vapor head above slurry 5050). The oxygen gascan be introduced into either an oxidation preprocessing chamber or intoan oxidation reactor (or into both), depending upon the configuration ofthe oxidative processing system. Typically, for example, the partialpressure of oxygen in the vapor above slurry 5050 is larger than theambient pressure of oxygen, and ranges from 0.5 bar to 35 bar, dependingupon the nature of the feedstock.

The oxygen gas can be introduced in pure form, or can be mixed with oneor more carrier gases. For example, in some embodiments, high-pressureair provides the oxygen in the vapor. In certain embodiments, oxygen gascan be supplied continuously to the vapor phase to ensure that aconcentration of oxygen in the vapor remains within certainpredetermined limits during processing of the feedstock. In someembodiments, oxygen gas can be introduced initially in sufficientconcentration to oxidize the feedstock, and then the feedstock can betransported to a closed, pressurized vessel (e.g., an oxidation reactor)for processing.

In certain embodiments, oxidizing agents 5160 can include nascent oxygen(e.g., oxygen radicals). Typically, nascent oxygen is produced as neededin an oxidation reactor or in a chamber in fluid communication with anoxidation reactor by one or more decomposition reactions. For example,in some embodiments, nascent oxygen can be produced from a reactionbetween NO and 02 in a gas mixture or in solution. In certainembodiments, nascent oxygen can be produced from decomposition of HOClin solution. Other methods by which nascent oxygen can be producedinclude via electrochemical generation in electrolyte solution, forexample.

In general, nascent oxygen is an efficient oxidizing agent due to therelatively high reactivity of the oxygen radical. However, nascentoxygen can also be a relatively selective oxidizing agent. For example,when lignocellulosic feedstock is treated with nascent oxygen, selectiveoxidation of lignin occurs in preference to the other components of thefeedstock such as cellulose. As a result, oxidation of feedstock withnascent oxygen provides a method for selective removal of the ligninfraction in certain feedstocks. Typically, nascent oxygen concentrationsof between about 0.5% and 5% of the dry weight of the feedstock are usedto effect efficient oxidation.

Without wishing to be bound by theory, it is believed that nascentoxygen reacts with lignocellulosic feedstock according to at least twodifferent mechanisms. In a first mechanism, nascent oxygen undergoes anaddition reaction with the lignin, resulting in partial oxidation of thelignin, which solubilizes the lignin in aqueous solution. As a result,the solubilized lignin can be removed from the rest of the feedstock viawashing. In a second mechanism, nascent oxygen disrupts butanecross-links and/or opens aromatic rings that are connected via thebutane cross-links. As a result, solubility of the lignin in aqueoussolution increases, facilitating separation of the lignin fraction fromthe remainder of the feedstock via washing.

In some embodiments, oxidizing agents 5160 include ozone (O3). The useof ozone can introduce several chemical handling considerations in theoxidation processing sequence. If heated too vigorously, an aqueoussolution of ozone can decompose violently, with potentially adverseconsequences for both human system operators and system equipment.Accordingly, ozone is typically generated in a thermally isolated,thick-walled vessel separate from the vessel that contains the feedstockslurry, and transported thereto at the appropriate process stage.

Without wishing to be bound by theory, it is believed that ozonedecomposes into oxygen and oxygen radicals, and that the oxygen radicals(e.g., nascent oxygen) are responsible for the oxidizing properties ofozone in the manner discussed above. Ozone typically preferentiallyoxidizes the lignin fraction in lignocellulosic materials, leaving thecellulose fraction relatively undisturbed.

Conditions for ozone-based oxidation of biomass feedstock generallydepend upon the nature of the biomass. For example, for cellulosicand/or lignocellulosic feedstocks, ozone concentrations of from 0.1 g/m3to 20 g/m3 of dry feedstock provide for efficient feedstock oxidation.Typically, the water content in slurry 5050 is between 10% by weight and80% by weight (e.g., between 40% by weight and 60% by weight). Duringozone-based oxidation, the temperature of slurry 5050 can be maintainedbetween 0° C. and 100° C. to avoid violent decomposition of the ozone.

In some embodiments, feedstock slurry 5050 can be treated with anaqueous, alkaline solution that includes one or more alkali and alkalineearth hydroxides such as sodium hydroxide, potassium hydroxide, andcalcium hydroxide, and then treated thereafter with an ozone-containinggas in an oxidation reactor. This process has been observed tosignificantly increase decomposition of the biomass in slurry 5050.Typically, for example, a concentration of hydroxide ions in thealkaline solution is between 0.001% and 10% by weight of slurry 5050.After the feedstock has been wetted via contact with the alkalinesolution, the ozone-containing gas is introduced into the oxidationreactor, where it contacts and oxidizes the feedstock.

Oxidizing agents 5160 can also include other substances. In someembodiments, for example, halogen-based oxidizing agents such aschlorine and oxychlorine agents (e.g., hypochlorite) can be introducedinto slurry 5050. In certain embodiments, nitrogen-containing oxidizingsubstances can be introduced into slurry 5050. Exemplarynitrogen-containing oxidizing substances include NO and NO2, forexample. Nitrogen-containing agents can also be combined with oxygen inslurry 5050 to create additional oxidizing agents. For example, NO andNO2 both combine with oxygen in slurry 5050 to form nitrate compounds,which are effective oxidizing agents for biomass feedstock. Halogen- andnitrogen-based oxidizing agents can, in some embodiments, causebleaching of the biomass feedstock, depending upon the nature of thefeedstock. The bleaching may be desirable for certain biomass-derivedproducts that are extracted in subsequent processing steps.

Other oxidizing agents can include, for example, various peroxyacids,peroxyacetic acids, persulfates, percarbonates, permanganates, osmiumtetroxide, and chromium oxides.

Following oxidation preprocessing step 5060, feedstock slurry 5050 isoxidized in step 5070. If oxidizing agents 5160 were added to slurry5050 in an oxidation reactor, then oxidation proceeds in the samereactor. Alternatively, if oxidizing agents 5160 were added to slurry5050 in a preprocessing chamber, then slurry 5050 is transported to anoxidation reactor via an in-line piping system. Once inside theoxidation reactor, oxidation of the biomass feedstock proceeds under acontrolled set of environmental conditions. Typically, for example, theoxidation reactor is a cylindrical vessel that is closed to the externalenvironment and pressurized. Both batch and continuous operation ispossible, although environmental conditions are typically easier tocontrol in in-line batch processing operations.

Oxidation of feedstock slurry 5050 typically occurs at elevatedtemperatures in the oxidation reactor. For example, the temperature ofslurry 5050 in the oxidation reactor is typically maintained above 100°C., in a range from 120° C. to 240° C. For many types of biomassfeedstock, oxidation is particularly efficient if the temperature ofslurry 5050 is maintained between 150° C. and 220° C. Slurry 5050 can beheating using a variety of thermal transfer devices. For example, insome embodiments, the oxidation reactor contacts a heating bath thatincludes oil or molten salts. In certain embodiments, a series of heatexchange pipes surround and contact the oxidation reactor, andcirculation of hot fluid within the pipes heats slurry 5050 in thereactor. Other heating devices that can be used to heat slurry 5050include resistive heating elements, induction heaters, and microwavesources, for example.

The residence time of feedstock slurry 5050 in the oxidation reactor canbe varied as desired to process the feedstock. Typically, slurry 5050spends from 1 minute to 60 minutes undergoing oxidation in the reactor.For relatively soft biomass material such as lignocellulosic matter, theresidence time in the oxidation reactor can be from 5 minutes to 30minutes, for example, at an oxygen pressure of between 3 and 12 bars inthe reactor, and at a slurry temperature of between 160° C. and 210° C.For other types of feedstock, however, residence times in the oxidationreactor can be longer, e.g., as long 48 hours. To determine appropriateresidence times for slurry 5050 in the oxidation reactor, aliquots ofthe slurry can be extracted from the reactor at specific intervals andanalyzed to determine concentrations of particular products of interestsuch as complex saccharides. Information about the increase inconcentrations of certain products in slurry 5050 as a function of timecan be used to determine residence times for particular classes offeedstock material.

In some embodiments, during oxidation of feedstock slurry 5050,adjustment of the slurry pH may be performed by introducing one or morechemical agents into the oxidation reactor. For example, in certainembodiments, oxidation occurs most efficiently in a pH range of about9-11. To maintain a pH in this range, agents such as alkali and alkalineearth hydroxides, carbonates, ammonia, and alkaline buffer solutions canbe introduced into the oxidation reactor.

Circulation of slurry 5050 during oxidation can be important to ensuresufficient contact between oxidizing agents 5160 and the feedstock.Circulation of the slurry can be achieved using a variety of techniques.For example, in some embodiments, a mechanical stirring apparatus thatincludes impeller blades or a paddle wheel can be implemented in theoxidation reactor. In certain embodiments, the oxidation reactor can bea loop reactor, in which the aqueous solvent in which the feedstock issuspended is simultaneously drained from the bottom of the reactor andrecirculated into the top of the reactor via pumping, thereby ensuringthat the slurry is continually re-mixed and does not stagnate within thereactor.

After oxidation of the feedstock is complete, the slurry is transportedto a separation apparatus where a mechanical separation step 5080occurs. Typically, mechanical separation step 5080 includes one or morestages of increasingly fine filtering of the slurry to mechanicallyseparate the solid and liquid constituents.

Liquid phase 5090 is separated from solid phase 5100, and the two phasesare processed independently thereafter. Solid phase 5100 can beoptionally undergo a dying step 5120 in a drying apparatus, for example.Drying step 5120 can include, for example, mechanically dispersing thesolid material onto a drying surface, and evaporating water from solidphase 5100 by gentle heating of the solid material. Following dryingstep 5120 (or, alternatively, without undergoing drying step 5120),solid phase 5100 is transported for further processing steps 5140.

Liquid phase 5090 can optionally undergo a drying step 5110 to reducethe concentration of water in the liquid phase. In some embodiments, forexample, drying step 5110 can include evaporation and/or distillationand/or extraction of water from liquid phase 5090 by gentle heating ofthe liquid. Alternatively, or in addition, one or more chemical dryingagents can be used to remove water from liquid phase 5090. Followingdrying step 5110 (or alternatively, without undergoing drying step5110), liquid phase 5090 is transported for further processing steps5130, which can include a variety of chemical and biological treatmentsteps such as chemical and/or enzymatic hydrolysis.

Drying step 5110 creates waste stream 5220, an aqueous solution that caninclude dissolved chemical agents such as acids and bases in relativelylow concentrations. Treatment of waste stream 5220 can include, forexample, pH neutralization with one or more mineral acids or bases.Depending upon the concentration of dissolved salts in waste stream5220, the solution may be partially de-ionized (e.g., by passing thewaste stream through an ion exchange system). Then, the wastestream—which includes primarily water—can be re-circulated into theoverall process (e.g., as water 5150), diverted to another process, ordischarged.

Typically, for lignocellulosic biomass feedstocks following separationstep 5070, liquid phase 5090 includes a variety of soluble poly- andoligosaccharides, which can then be separated and/or reduced tosmaller-chain saccharides via further processing steps. Solid phase 5100typically includes primarily cellulose, for example, with smalleramounts of hemicellulose- and lignin-derived products.

In some embodiments, oxidation can be carried out at elevatedtemperature in a reactor such as a pyrolysis chamber. For example,referring again to FIG. 17, feedstock materials can be oxidized infilament pyrolyzer 1712. In a typical usage, an oxidizing carrier gas,e.g., air or an air/argon blend, transverses through a sample holder1713 while the resistive heating element is rotated and heated to adesired temperature, e.g., 325° C. After an appropriate time, e.g., 5 to10 minutes, the oxidized material is emptied from the sample holder. Thesystem shown in FIG. 17 can be scaled and made continuous. For example,rather than a wire as the heating member, the heating member can be anauger screw. Material can continuously fall into the sample holder,striking a heated screw that pyrolizes the material. At the same time,the screw can push the oxidized material out of the sample holder toallow for the entry of fresh, unoxidized material.

Feedstock materials can also be oxidized in any of the pyrolysis systemsshown in FIGS. 18-20 and described above in the Pyrolysis Systemssection.

Referring again to FIG. 21, feedstock materials can be rapidly oxidizedby coating a tungsten filament 150, together with an oxidant, such as aperoxide, with the desired cellulosic material while the material ishoused in a vacuum chamber 151. To affect oxidation, current is passedthrough the filament, which causes a rapid heating of the filament for adesired time. Typically, the heating is continued for seconds beforeallowing the filament to cool. In some embodiments, the heating isperformed a number of times to effect the desired amount of oxidation.

Referring again to FIG. 12, in some embodiments, feedstock materials canbe oxidized with the aid of sound and/or cavitation. Generally, toeffect oxidation, the materials are sonicated in an oxidizingenvironment, such as water saturated with oxygen or another chemicaloxidant, such as hydrogen peroxide.

Referring again to FIGS. 9 and 10, in certain embodiments, ionizingradiation is used to aid in the oxidation of feedstock materials.Generally, to effect oxidation, the materials are irradiated in anoxidizing environment, such as air or oxygen. For example, gammaradiation and/or electron beam radiation can be employed to irradiatethe materials.

Other Processes

Steam explosion can be used alone without any of the processes describedherein, or in combination with any of the processes described herein.

FIG. 23 shows an overview of the entire process of converting a fibersource 400 into a product 450, such as ethanol, by a process thatincludes shearing and steam explosion to produce a fibrous material 401,which is then hydrolyzed and converted, e.g., fermented, to produce theproduct. The fiber source can be transformed into the fibrous material401 through a number of possible methods, including at least oneshearing process and at least one steam explosion process.

For example, one option includes shearing the fiber source, followed byoptional screening step(s) and optional additional shearing step(s) toproduce a sheared fiber source 402, which can then be steam exploded toproduce the fibrous material 401. The steam explosion process isoptionally followed by a fiber recovery process to remove liquids or the“liquor” 404, resulting from the steam exploding process. The materialresulting from steam exploding the sheared fiber source may be furthersheared by optional additional shearing step(s) and/or optionalscreening step(s).

In another method, the fibrous material 401 is first steam exploded toproduce a steam exploded fiber source 410. The resulting steam explodedfiber source is then subjected to an optional fiber recovery process toremove liquids, or the liquor. The resulting steam exploded fiber sourcecan then be sheared to produce the fibrous material. The steam explodedfiber source can also be subject to one or more optional screening stepsand/or one or more optional additional shearing steps. The process ofshearing and steam exploding the fiber source to produce the sheared andsteam exploded fibrous material will be further discussed below.

The fiber source can be cut into pieces or strips of confetti materialprior to shearing or steam explosion. The shearing processes can takeplace with the material in a dry state (e.g., having less than 0.25percent by weight absorbed water), a hydrated state, or even while thematerial is partially or fully submerged in a liquid, such as water orisopropanol. The process can also optimally include steps of drying theoutput after steam exploding or shearing to allow for additional stepsof dry shearing or steam exploding. The steps of shearing, screening,and steam explosion can take place with or without the presence ofvarious chemical solutions.

In a steam explosion process, the fiber source or the sheared fibersource is contacted with steam under high pressure, and the steamdiffuses into the structures of the fiber source (e.g., thelignocellulosic structures). The steam then condenses under highpressure thereby “wetting” the fiber source. The moisture in the fibersource can hydrolyze any acetyl groups in the fiber source (e.g., theacetyl groups in the hemicellulose fractions), forming organic acidssuch as acetic and monic acids. The acids, in turn, can catalyze thedepolymerization of hemicellulose, releasing xylan and limited amountsof glucan. The “wet” fiber source (or sheared fiber source, etc.) isthen “exploded” when the pressure is released. The condensed moistureinstantaneously evaporates due to the sudden decrease in pressure andthe expansion of the water vapor exerts a shear force upon the fibersource (or sheared fiber source, etc.). A sufficient shear force willcause the mechanical breakdown of the internal structures (e.g., thelignocellulosic structures) of the fiber source.

The sheared and steam exploded fibrous material is then converted into auseful product, such as ethanol. In some embodiments, the fibrousmaterial is converted into a fuel. One method of converting the fibrousmaterial into a fuel is by hydrolysis to produce fermentable sugars,412, which are then fermented to produce the product. Other known andunknown methods of converting fibrous materials into fuels may also beused.

In some embodiments, prior to combining the microorganism, the shearedand steam exploded fibrous material 401 is sterilized to kill anycompeting microorganisms that may be on the fibrous material. Forexample, the fibrous material can be sterilized by exposing the fibrousmaterial to radiation, such as infrared radiation, ultravioletradiation, or an ionizing radiation, such as gamma radiation. Themicroorganisms can also be killed using chemical sterilants, such asbleach (e.g., sodium hypochlorite), chlorhexidine, or ethylene oxide.

One method to hydrolyze the sheared and steam exploded fibrous materialis by the use of cellulases. Cellulases are a group of enzymes that actsynergistically to hydrolyze cellulose. Commercially availableAccellerase® 1000 enzyme complex, which contains a complex of enzymesthat reduces lignocellulosic biomass into fermentable sugars, can alsobe used.

According to current understanding, the components of cellulase includeendoglucanases, exoglucanases (cellobiohydrolases), and b-glucosidases(cellobiases). Synergism between the cellulase components exists whenhydrolysis by a combination of two or more components exceeds the sum ofthe activities expressed by the individual components. The generallyaccepted mechanism of a cellulase system (particularly of T.longibrachiatum) on crystalline cellulose is: endoglucanase hydrolyzesinternal-1,4-glycosidic bonds of the amorphous regions, therebyincreasing the number of exposed nonreducing ends. Exoglucanases thencleave off cellobiose units from the nonreducing ends, which in turn arehydrolyzed to individual glucose units by b-glucosidases. There areseveral configurations of both endo- and exo-glucanases differing instereospecificities. In general, the synergistic action of thecomponents in various configurations is required for optimum cellulosehydrolysis. Cellulases, however, are more inclined to hydrolyze theamorphous regions of cellulose. A linear relationship betweencrystallinity and hydrolysis rates exists whereby higher crystallinityindices correspond to slower enzyme hydrolysis rates. Amorphous regionsof cellulose hydrolyze at twice the rate of crystalline regions. Thehydrolysis of the sheared and steam exploded fibrous material may beperformed by any hydrolyzing biomass process.

Steam explosion of biomass sometimes causes the formation ofby-products, e.g., toxicants, that are inhibitory to microbial andenzymatic activities. The process of converting the sheared and steamexploded fibrous material into a fuel can therefore optionally includean overliming step prior to fermentation to precipitate some of thetoxicants. For example, the pH of the sheared and steam exploded fibrousmaterial may be raised to exceed the pH of 10 by adding calciumhydroxide (Ca(OH)2) followed by a step of lowering the pH to about 5 byadding H2 S04. The overlimed fibrous material may then be used as iswithout the removal of precipitates. As shown in FIG. 23, the optionaloverliming step occurs just prior to the step of hydrolysis of thesheared and steam exploded fibrous material, but it is also contemplatedto perform the overliming step after the hydrolysis step and prior tothe fermenting step.

FIG. 24 depicts an example of a steam explosion apparatus 460. The steamexplosion apparatus 460 includes a reaction chamber 462, in which thefiber source and/or the fibrous material is placed through a fibersource inlet 464. Closing fiber source inlet valve 465 seals thereaction chamber. The reaction chamber further includes a pressurizedsteam inlet 466 that includes a steam valve 467. The reaction chamberfurther includes an explosive depressurization outlet 468 that includesan outlet valve 469 in communication with the cyclone 470 through theconnecting pipe 472. Once the reaction chamber contains the fiber sourceand/or sheared fiber source and is sealed by closing valves 465, 467 and469, steam is delivered into the reaction chamber 462 by opening thesteam inlet valve 467 allowing steam to travel through steam inlet 466.Once the reaction chamber reaches target temperature, which can takeabout 20-60 seconds, the holding time begins. The reaction chamber isheld at the target temperature for the desired holding time, whichtypically lasts from about 10 seconds to 5 minutes. At the end of theholding time period, outlet valve is opened to allow for explosivedepressurization to occur. The process of explosive depressurizationpropels the contents of the reaction chamber 462 out of the explosivedepressurization outlet 468, through the connecting pipe 472, and intothe cyclone 470. The steam exploded fiber source or fibrous materialthen exits the cyclone in a sludge form into the collection bin 474 asmuch of the remaining steam exits the cyclone into the atmospherethrough vent 476. The steam explosion apparatus further includes washoutlet 478 with wash outlet valve 479 in communication with connectingpipe 472. The wash outlet valve 479 is closed during the use of thesteam explosion apparatus 460 for steam explosion, but opened during thewashing of the reaction chamber 462.

The target temperature of the reaction chamber 462 is preferably between180 and 240 degrees Celsius or between 200 and 220 degrees Celsius. Theholding time is preferably between 10 seconds and 30 minutes, or between30 seconds and 10 minutes, or between 1 minute and 5 minutes.

Because the steam explosion process results in a sludge of steamexploded fibrous material, the steam exploded fibrous material mayoptionally include a fiber recovery process where the “liquor” isseparated from the steam exploded fibrous material. This fiber recoverystep is helpful in that it enables further shearing and/or screeningprocesses and can allow for the conversion of the fibrous material intofuel. The fiber recovery process occurs through the use of a mesh clothto separate the fibers from the liquor. Further drying processes canalso be included to prepare the fibrous material or steam exploded fibersource for subsequent processing.

Combined Irradiating, Pyrolizing, Sonicating, and/or Oxidizing Devices

In some embodiments, it may be advantageous to combine two or moreseparate irradiation, sonication, pyrolyzation, and/or oxidation devicesinto a single hybrid machine. Using such a hybrid machine, multipleprocesses may be performed in close juxtaposition or evensimultaneously, with the benefit of increasing pretreatment throughputand potential cost savings.

For example, consider the electron beam irradiation and sonicationprocesses. Each separate process is effective in lowering the meanmolecular weight of cellulosic material by an order of magnitude ormore, and by several orders of magnitude when performed serially.

Both irradiation and sonication processes can be applied using a hybridelectron beam/sonication device as is illustrated in FIG. 25. Hybridelectron beam/sonication device 2500 is pictured above a shallow pool(depth 3-5 cm) of a slurry of cellulosic material 2550 dispersed in anaqueous, oxidant medium, such as hydrogen peroxide or carbamideperoxide. Hybrid device 2500 has an energy source 2510, which powersboth electron beam emitter 2540 and sonication horns 2530.

Electron beam emitter 2540 generates electron beams, which pass throughan electron beam aiming device 2545 to impact the slurry 2550 containingcellulosic material. The electron beam-aiming device can be a scannerthat sweeps a beam over a range of up to about 6 feet in a directionapproximately parallel to the surface of the slurry 2550.

On either side of the electron beam emitter 2540 are sonication horns2530, which deliver ultrasonic wave energy to the slurry 2550. Thesonication horns 2530 end in a detachable endpiece 2535 that is incontact with the slurry 2550.

The sonication horns 2530 are at risk of damage from long-term residualexposure to the electron beam radiation. Thus, the horns can beprotected with a standard shield 2520, e.g., made of lead or aheavy-metal-containing alloy such as Lipowitz metal, which is imperviousto electron beam radiation. Precautions must be taken, however, toensure that the ultrasonic energy is not affected by the presence of theshield. The detachable endpieces 2535, which are constructed of the samematerial and attached to the horns 2530, are in contact with thecellulosic material 2550 during processing and are expected to bedamaged. Accordingly, the detachable endpieces 2535 are constructed tobe easily replaceable.

A further benefit of such a simultaneous electron beam and ultrasoundprocess is that the two processes have complementary results. Withelectron beam irradiation alone, an insufficient dose may result incross-linking of some of the polymers in the cellulosic material, whichlowers the efficiency of the overall depolymerization process. Lowerdoses of electron beam irradiation and/or ultrasound radiation may alsobe used to achieve a similar degree of depolymerization as that achievedusing electron beam irradiation and sonication separately. An electronbeam device can also be combined with one or more of high-frequency,rotor-stator devices, which can be used as an alternative to ultrasonicsonication devices.

Further combinations of devices are also possible. For example, anionizing radiation device that produces gamma radiation emitted from,e.g., 6° C. pellets, can be combined with an electron beam source and/oran ultrasonic wave source. Shielding requirements may be more stringentin this case.

The radiation devices for pretreating biomass discussed above can alsobe combined with one or more devices that perform one or more pyrolysisprocessing sequences. Such a combination may again have the advantage ofhigher throughput. Nevertheless, caution must be observed, as there maybe conflicting requirements between some radiation processes andpyrolysis. For example, ultrasonic radiation devices may require thefeedstock be immersed in a liquid oxidizing medium. On the other hand,as discussed previously, it may be advantageous for a sample offeedstock undergoing pyrolysis to be of a particular moisture content.In this case, the new systems automatically measure and monitor for aparticular moisture content and regulate the same. Further, some or allof the above devices, especially the pyrolysis device, can be combinedwith an oxidation device as discussed previously.

Primary Processes

Fermentation

Generally, various microorganisms can produce a number of usefulproducts, such as a fuel, by operating on, e.g., fermenting thepretreated biomass materials. For example, fermentation or otherprocesses can produce alcohols, organic acids, hydrocarbons, hydrogen,proteins or mixtures of any of these materials.

The microorganism can be a natural microorganism or an engineeredmicroorganism. For example, the microorganism can be a bacterium, e.g.,a cellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist,e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold.When the organisms are compatible, mixtures of organisms can beutilized.

To aid in the breakdown of the materials that include the cellulose, oneor more enzymes, e.g., a cellulolytic enzyme can be utilized. In someembodiments, the materials that include the cellulose are first treatedwith the enzyme, e.g., by combining the material and the enzyme in anaqueous solution. This material can then be combined with themicroorganism. In other embodiments, the materials that include thecellulose, the one or more enzymes and the microorganism are combined atthe concurrently, e.g., by combining in an aqueous solution.

Also, to aid in the breakdown of the materials that include thecellulose, the materials can be treated post irradiation with heat, achemical (e.g., mineral acid, base or a strong oxidizer such as sodiumhypochlorite), and/or an enzyme.

During the fermentation, sugars released from cellulolytic hydrolysis orthe saccharification step, are fermented to, e.g., ethanol, by afermenting microorganism such as yeast. Suitable fermentingmicroorganisms have the ability to convert carbohydrates, such asglucose, xylose, arabinose, mannose, galactose, oligosaccharides orpolysaccharides into fermentation products. Fermenting microorganismsinclude strains of the genus Saccharomyces spp. e.g., Saccharomycescerevisiae (baker's yeast), Saccharomyces distaticus, Saccharomycesuvarum; the genus Kluyveromyces, e.g., species Kluyveromyces marxianus,Kluyveromyces fragilis; the genus Candida, e.g., Candidapseudotropicalis, and Candida brassicae, Pichia stipitis (a relative ofCandida shehatae, the genus Clavispora, e.g., species Clavisporalusitaniae and Clavispora opuntiae the genus Pachysolen, e.g., speciesPachysolen tannophilus, the genus Bretannomyces, e.g., speciesBretannomyces clausenii (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212).

Commercially available yeast include, for example, Red Star®/LesaffreEthanol Red (available from Red Star/Lesaffre, USA) FALI® (availablefrom Fleischmann's Yeast, a division of Burns Philip Food Inc., USA),SUPERSTART® (available from Alltech), GERT STRAND® (available from GertStrand AB, Sweden) and FERMOL® (available from DSM Specialties).

Bacteria that can ferment biomass to ethanol and other products include,e.g., Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996,supra). Leschine et al. (International Journal of Systematic andEvolutionary Microbiology 2002, 52, 1155-1160) isolated an anaerobic,mesophilic, cellulolytic bacterium from forest soil, Clostridiumphytofermentans sp. nov., which converts cellulose to ethanol.

Fermentation of biomass to ethanol and other products may be carried outusing certain types of thermophilic or genetically engineeredmicroorganisms, such Thermoanaerobacter species, including T. mathranii,and yeast species such as Pichia species. An example of a strain of T.mathranii is A3M4 described in Sonne-Hansen et al. (Applied Microbiologyand Biotechnology 1993, 38, 537-541) or Ahring et al. (Arch. Microbial.1997, 168, 114-119).

Yeast and Zymomonas bacteria can be used for fermentation or conversion.The optimum pH for yeast is from about pH 4 to 5, while the optimum pHfor Zymomonas is from about pH 5 to 6. Typical fermentation times areabout 24 to 96 hours with temperatures in the range of 26° C. to 40° C.,however thermophilic microorganisms prefer higher temperatures.

Enzymes which break down biomass, such as cellulose, to lower molecularweight carbohydrate-containing materials, such as glucose, duringsaccharification are referred to as cellulolytic enzymes or cellulase.These enzymes may be a complex of enzymes that act synergistically todegrade crystalline cellulose. Examples of cellulolytic enzymes include:endoglucanases, cellobiohydrolases, and cellobiases (-glucosidases). Acellulosic substrate is initially hydrolyzed by endoglucanases at randomlocations producing oligomeric intermediates. These intermediates arethen substrates for exo-splitting glucanases such as cellobiohydrolaseto produce cellobiose from the ends of the cellulose polymer. Cellobioseis a water-soluble-1,4-linked dimer of glucose. Finally, cellobiasecleaves cellobiose to yield glucose.

A cellulase is capable of degrading biomass and may be of fungal orbacterial ongm. Suitable enzymes include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,Chrysosporium and Trichoderma, and include species of Humicola,Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium,Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, e.g., EP458162), especially those produced by a strain selected from the speciesHumicola insolens (reclassified as Scytalidium thermophilum, see, e.g.,U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysporum,Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris,Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremoniumbrachypenium, Acremonium dichromosporum, Acremonium obclavatum,Acremonium pinkertoniae, Acremonium roseogriseum, Acremoniumincoloratum, and Acremonium furatum; preferably from the speciesHumicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthorathermophila CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65,Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71,Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS146.62, and Acremonium furatum CBS 299.70H. Cellulolytic enzymes mayalso be obtained from Chrysosporium, preferably a strain ofChrysosporium lucknowense. Additionally, Trichoderma (particularlyTrichoderma viride, Trichoderma reesei, and Trichoderma koningii),alkalophilic Bacillus (see, for example, U.S. Pat. No. 3,844,890 and EP458162), and Streptomyces (see, e.g., EP 458162) may be used. Thebacterium, Saccharophagus degradans, produces a mixture of enzymescapable of degrading a range of cellulosic materials and may also beused in this process.

Anaerobic cellulolytic bacteria have also been isolated from soil, e.g.,a novel cellulolytic species of Clostiridium, Clostridiumphytofermentans sp. nov. (see Leschine et. al, International Journal ofSystematic and Evolutionary Microbiology (2002), 52, 1155-1160).

Cellulolytic enzymes using recombinant technology can also be used (see,e.g., WO 2007/071818 and WO 2006/110891).

The cellulolytic enzymes used can be produced by fermentation of theabove-noted microbial strains on a nutrient medium containing suitablecarbon and nitrogen sources and inorganic salts, using procedures knownin the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection).

Temperature ranges and other conditions suitable for growth andcellulase production are known in the art (see, e.g., Bailey, J. E., andOllis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, NY, 1986).

Treatment of cellulose with cellulase is usually carried out attemperatures between 30° C. and 65° C. Cellulases are active over arange of pH of about 3 to 7. A saccharification step may last up to 120hours. The cellulase enzyme dosage achieves a sufficiently high level ofcellulose conversion. For example, an appropriate cellulase dosage istypically between 5.0 and 50 Filter Paper Units (FPU or IU) per gram ofcellulose. The FPU is a standard measurement and is defined and measuredaccording to Ghose (1987, Pure and Appl. Chem. 59:257-268).

Mobile fermentors can be utilized, as described in U.S. ProvisionalPatent Application Ser. No. 60/832,735, now Published InternationalApplication No. WO 2008/011598.

Gasification

In addition to using pyrolysis for pre-treatment of feedstock, pyrolysiscan also be used to process pre-treated feedstock to extract usefulmaterials. In particular, a form of pyrolysis known as gasification canbe employed to generate fuel gases along with various other gaseous,liquid, and solid products. To perform gasification, the pre-treatedfeedstock is introduced into a pyrolysis chamber and heated to a hightemperature, typically 700° C. or more. The temperature used dependsupon a number of factors, including the nature of the feedstock and thedesired products.

Quantities of oxygen (e.g., as pure oxygen gas and/or as air) and steam(e.g., superheated steam) are also added to the pyrolysis chamber tofacilitate gasification. These compounds react with carbon-containingfeedstock material in a multiple-step reaction to generate a gas mixturecalled synthesis gas (or “syngas”). Essentially, during gasification, alimited amount of oxygen is introduced into the pyrolysis chamber toallow some feedstock material to combust to form carbon monoxide andgenerate process heat. The process heat can then be used to promote asecond reaction that converts additional feedstock material to hydrogenand carbon monoxide.

In a first step of the overall reaction, heating the feedstock materialproduces a char that can include a wide variety of differenthydrocarbon-based species. Certain volatile materials can be produced(e.g., certain gaseous hydrocarbon materials), resulting in a reductionof the overall weight of the feedstock material. Then, in a second stepof the reaction, some of the volatile material that is produced in thefirst step reacts with oxygen in a combustion reaction to produce bothcarbon monoxide and carbon dioxide. The combustion reaction releasesheat, which promotes the third step of the reaction. In the third step,carbon dioxide and steam (e.g., water) react with the char generated inthe first step to form carbon monoxide and hydrogen gas. Carbon monoxidecan also react with steam, in a water gas shift reaction, to form carbondioxide and further hydrogen gas.

Gasification can be used as a primary process to generate productsdirectly from pre-treated feedstock for subsequent transport and/orsale, for example. Alternatively, or in addition, gasification can beused as an auxiliary process for generating fuel for an overallprocessing system. The hydrogen-rich syngas that is generated via thegasification process can be burned, for example, to generate electricityand/or process heat that can be directed for use at other locations inthe processing system. As a result, the overall processing system can beat least partially self-sustaining. A number of other products,including pyrolysis oils and gaseous hydrocarbon-based substances, canalso be obtained during and/or following gasification; these can beseparated and stored or transported as desired.

A variety of different pyrolysis chambers are suitable for gasificationof pre-treated feedstock, including the pyrolysis chambers disclosedherein. In particular, fluidized bed reactor systems, in which thepre-treated feedstock is fluidized in steam and oxygen/air, providerelatively high throughput and straightforward recovery of products.Solid char that remains following gasification in a fluidized bed system(or in other pyrolysis chambers) can be burned to generate additionalprocess heat to promote subsequent gasification reactions.

Post-Processing

Distillation

After fermentation, the resulting fluids can be distilled using, forexample, a “beer column” to separate ethanol and other alcohols from themajority of water and residual solids. The vapor exiting the beer columncan be, e.g., 35% by weight ethanol and can be fed to a rectificationcolumn. A mixture of nearly azeotropic (92.5%) ethanol and water fromthe rectification column can be purified to pure (99.5%) ethanol usingvapor-phase molecular sieves. The beer column bottoms can be sent to thefirst effect of a three-effect evaporator. The rectification columnreflux condenser can provide heat for this first effect. After the firsteffect, solids can be separated using a centrifuge and dried in a rotarydryer. A portion (25%) of the centrifuge effluent can be recycled tofermentation and the rest sent to the second and third evaporatoreffects. Most of the evaporator condensate can be returned to theprocess as fairly clean condensate with a small portion split off towaste water treatment to prevent build-up of low-boiling compounds.

Waste Water Treatment

Wastewater treatment is used to minimize makeup water requirements ofthe plant by treating process water for reuse within the plant.Wastewater treatment can also produce fuel (e.g., sludge and biogas)that can be used to improve the overall efficiency of the ethanolproduction process. For example, as described in further detail below,sludge and biogas can be used to create steam and electricity that canbe used in various plant processes.

Wastewater is initially pumped through a screen (e.g., a bar screen) toremove large particles, which are collected in a hopper. In someembodiments, the large particles are sent to a landfill. Additionally oralternatively, the large particles are burned to create steam and/orelectricity as described in further detail below. In general, thespacing on the bar screen is between ¼ inch to 1 inch spacing (e.g., ½inch spacing).

The wastewater then flows to an equalization tank, where the organicconcentration of the wastewater is equalized during a retention time. Ingeneral, the retention time is between 8 hours and 36 hours (e.g., 24hours). A mixer is disposed within the tank to stir the contents of thetank. In some embodiments, a plurality of mixers disposed throughout thetank are used to stir the contents of the tank. In certain embodiments,the mixer substantially mixes the contents of the equalization tank suchthat conditions (e.g., wastewater concentration and temperature)throughout the tank are uniform.

A first pump moves water from the equalization tank through aliquid-to-liquid heat exchanger. The heat exchanger is controlled (e.g.,by controlling the flow rate of fluid through the heat exchanger) suchthat wastewater exiting the heat exchanger is at a desired temperaturefor anaerobic treatment. For example, the desired temperature foranaerobic treatment can be between 40° C. to 60° C.

After exiting the heat exchanger, the wastewater enters one or moreanaerobic reactors. In some embodiments, the concentration of sludge ineach anaerobic reactor is the same as the overall concentration ofsludge in the wastewater. In other embodiments, the anaerobic reactorhas a higher concentration of sludge than the overall concentration ofsludge in the wastewater.

A nutrient solution containing nitrogen and phosphorus is metered intoeach anaerobic reactor containing wastewater. The nutrient solutionreacts with the sludge in the anaerobic reactor to produce biogas whichcan contain 50% methane and have a heating value of approximately 12,000British thermal units, or Btu, per pound). The biogas exits eachanaerobic reactor through a vent and flows into a manifold, where aplurality of biogas streams are combined into a single stream. Acompressor moves the stream of biogas to a boiler or a combustion engineas described in further detail below. In some embodiments, thecompressor also moves the single stream of biogas through adesulphurization catalyst. Additionally or alternatively, the compressorcan move the single stream of biogas through a sediment trap.

A second pump moves anaerobic effluent from the anaerobic reactors toone or more aerobic reactors (e.g., activated sludge reactors). Anaerator is disposed within each aerobic reactor to mix the anaerobiceffluent, sludge, and oxygen (e.g., oxygen contained in air). Withineach aerobic reactor, oxidation of cellular material in the anaerobiceffluent produces carbon dioxide, water, and ammonia.

Aerobic effluent moves (e.g., via gravity) to a separator, where sludgeis separated from treated water. Some of the sludge is returned to theone or more aerobic reactors to create an elevated sludge concentrationin the aerobic reactors, thereby facilitating the aerobic breakdown ofcellular material in the wastewater. A conveyor removes excess sludgefrom the separator. As described in further detail below, the excesssludge is used as fuel to create steam and/or electricity.

The treated water is pumped from the separator to a settling tank.Solids dispersed throughout the treated water settle to the bottom ofthe settling tank and are subsequently removed. After a settling period,treated water is pumped from the settling tank through a fine filter toremove any additional solids remaining in the water. In someembodiments, chlorine is added to the treated water to kill pathogenicbacteria. In some embodiments, one or more physical-chemical separationtechniques are used to further purify the treated water. For example,treated water can be pumped through a carbon adsorption reactor. Asanother example, treated water can pumped through a reverse osmosisreactor. In the processes disclosed herein, whenever water is used inany process, it may be grey water, e.g., municipal grey water, or blackwater. In some embodiments, the grey or black water is sterilized priorto use. Sterilization may be accomplished by any desired technique, forexample by irradiation, steam, or chemical sterilization.

Waste Combustion

The production of alcohol from biomass can result in the production ofvarious by-product streams useful for generating steam and electricityto be used in other parts of the plant. For example, steam generatedfrom burning by-product streams can be used in the distillation process.As another example, electricity generated from burning by-productstreams can be used to power electron beam generators and ultrasonictransducers used in pretreatment.

The by-products used to generate steam and electricity are derived froma number of sources throughout the process. For example, anaerobicdigestion of wastewater produces a biogas high in methane and a smallamount of waste biomass (sludge). As another example, post-distillatesolids (e.g., unconverted lignin, cellulose, and hemicellulose remainingfrom the pretreatment and primary processes) can be used as a fuel.

The biogas is diverted to a combustion engine connected to an electricgenerator to produce electricity. For example, the biogas can be used asa fuel source for a spark-ignited natural gas engine. As anotherexample, the biogas can be used as a fuel source for a direct-injectionnatural gas engine. As another example, the biogas can be used as a fuelsource for a combustion turbine. Additionally or alternatively, thecombustion engine can be configured in a cogeneration configuration. Forexample, waste heat from the combustion engines can be used to providehot water or steam throughout the plant.

The sludge, and post-distillate solids are burned to heat water flowingthrough a heat exchanger. In some embodiments, the water flowing throughthe heat exchanger is evaporated and superheated to steam. In certainembodiments, the steam is used in the pretreatment rector and in heatexchange in the distillation and evaporation processes. Additionally oralternatively, the steam expands to power a multi-stage steam turbineconnected to an electric generator. Steam exiting the steam turbine iscondensed with cooling water and returned to the heat exchanger forreheating to steam. In some embodiments, the flow rate of water throughthe heat exchanger is controlled to obtain a target electricity outputfrom the steam turbine connected to an electric generator. For example,water can be added to the heat exchanger to ensure that the steamturbine is operating above a threshold condition (e.g., the turbine isspinning fast enough to turn the electric generator).

While certain embodiments have been described, other embodiments arepossible.

As an example, while the biogas is described as being diverted to acombustion engine connected to an electric generator, in certainembodiments, the biogas can be passed through a fuel reformer to producehydrogen. The hydrogen is then converted to electricity through a fuelcell.

As another example, while the biogas is described as being burned apartfrom the sludge and post-distillate solids, in certain embodiments, allof the waste by-products can be burned together to produce steam.

Products I Co-Products

Alcohols

The alcohol produced can be a monohydroxy alcohol, e.g., ethanol, or apolyhydroxy alcohol, e.g., ethylene glycol or glycerin. Examples ofalcohols that can be produced include methanol, ethanol, propanol,isopropanol, butanol, e.g., n-, sec- or t-butanol, ethylene glycol,propylene glycol, 1,4-butane diol, glycerin or mixtures of thesealcohols.

Each of the alcohols produced by the plant have commercial value asindustrial feedstock. For example, ethanol can be used in themanufacture of varnishes and perfume. As another example, methanol canbe used as a solvent used as a component in windshield wiper fluid. Asstill another example, butanol can be used in plasticizers, resins,lacquers, and brake fluids.

Bioethanol produced by the plant is valuable as an ingredient used inthe food and beverage industry. For example, the ethanol produced by theplant can be purified to food grade alcohol and used as a primaryingredient in the alcoholic beverages.

Bioethanol produced by the plant also has commercial value as atransportation fuel. The use of ethanol as a transportation fuel can beimplemented with relatively little capital investment from sparkignition engine manufacturers and owners (e.g., changes to injectiontiming, fuel-to-air ratio, and components of the fuel injection system).Many automotive manufacturers currently offer flex-fuel vehicles capableof operation on ethanol/gasoline blends up to 85% ethanol by volume(e.g., standard equipment on a Chevy Tahoe 4×4).

Bioethanol produced by this plant can be used as an engine fuel toimprove environmental and economic conditions beyond the location of theplant. For example, ethanol produced by this plant and used as a fuelcan reduce greenhouse gas emissions from manmade sources (e.g.,transportation sources). As another example, ethanol produced by thisplant and used as an engine fuel can also displace consumption ofgasoline refined from oil.

Bioethanol has a greater octane number than conventional gasoline and,thus, can be used to improve the performance (e.g., allow for highercompression ratios) of spark ignition engines. For example, smallamounts (e.g., 10% by volume) of ethanol can be blended with gasoline toact as an octane enhancer for fuels used in spark ignition engines. Asanother example, larger amounts (e.g., 85% by volume) of ethanol can beblended with gasoline to further increase the fuel octane number anddisplace larger volumes of gasoline.

Bioethanol strategies are discussed, e.g., by DiPardo in Journal ofOutlook for Biomass Ethanol Production and Demand (EIA Forecasts), 2002;Sheehan in Biotechnology Progress, 15:8179, 1999; Martin in EnzymeMicrobes Technology, 31:274, 2002; Greer in BioCycle, 61-65, April 2005;Lynd in Microbiology and Molecular Biology Reviews, 66:3, 506-577, 2002;Ljungdahl et al. in U.S. Pat. No. 4,292,406; and Bellamy in U.S. Pat.No. 4,094,742.

Organic Acids

The organic acids produced can include monocarboxylic acids orpolycarboxylic acids. Examples of organic acids include formic acid,acetic acid, propionic acid, butyric acid, valeric acid, caproic,palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid,glutaric acid, oleic acid, linoleic acid, glycolic acid, lactic acid,y-hydroxybutyric acid or mixtures of these acids.

Food Products

In some embodiments, all or a portion of the fermentation process can beinterrupted before the cellulosic material is completely converted toethanol. The intermediate fermentation products include highconcentrations of sugar and carbohydrates. These intermediatefermentation products can be used in preparation of food for human oranimal consumption. In some embodiments, irradiation pretreatment of thecellulosic material will render the intermediate fermentation productssterile (e.g., fit for human consumption). In some embodiments, theintermediate fermentation products will require post-processing prior touse as food. For example, a dryer can be used to remove moisture fromthe intermediate fermentation products to facilitate storage, handling,and shelf life. Additionally or alternatively, the intermediatefermentation products can be ground to a fine particle size in astainless-steel laboratory mill to produce a flour-like substance.

Animal Feed

Distillers grains and solubles can be converted into a valuablebyproduct of the distillation-dehydration process. After thedistillation-dehydration process, distillers grains and solubles can bedried to improve the ability to store and handle the material. Theresulting dried distillers grains and solubles (DDGS) is low in starch,high in fat, high in protein, high in fiber, and high in phosphorous.Thus, for example, DDGS can be valuable as a source of animal feed(e.g., as a feed source for dairy cattle). DDGS can be subsequentlycombined with nutritional additives to meet specific dietaryrequirements of specific categories of animals (e.g., balancingdigestible lysine and phosphorus for swine diets).

Pharmaceuticals

The pretreatment processes discussed above can be applied to plants withmedicinal properties. In some embodiments, sonication can stimulatebioactivity and/or bioavailability of the medicinal components of plantswith medicinal properties. Additionally or alternatively, irradiationstimulates bioactivity and/or bioavailability of the medicinalcomponents of plants with medicinal properties. For example, sonicationand irradiation can be combined in the pretreatment of willow bark tostimulate the production of salicin.

Nutriceuticals

In some embodiments, intermediate fermentation products (e.g., productsthat include high concentrations of sugar and carbohydrates) can besupplemented to create a nutriceutical. For example, intermediatefermentation products can be supplemented with calcium to create anutriceutical that provides energy and helps improve or maintain bonestrength.

Co-Products

Lignin Residue

As described above, lignin-containing residues from primary andpretreatment processes has value as a high/medium energy fuel and can beused to generate power and steam for use in plant processes. However,such lignin residues are a new type of solids fuel and there is littledemand for it outside of the plant boundaries, and the costs of dryingit for transportation only subtract from its potential value. In somecases, gasification of the lignin residues can convert it to ahigher-value product with lower cost.

Other Co-Products

Cell matter, furfural, and acetic acid have been identified as potentialco-products of biomass-to-fuel processing facilities. Interstitial cellmatter could be valuable, but might require significant purification.Markets for furfural and acetic acid are in place, although it isunlikely that they are large enough to consume the output of a fullycommercialized lignocellulose-to-ethanol industry.

EXAMPLES

The following Examples are intended to illustrate, and do not limit theteachings of this disclosure.

Example 1 Preparation of Fibrous Material from Polycoated Paper

A 1500 pound skid of virgin, half-gallon juice cartons made ofun-printed polycoated white Kraft board having a bulk density of 20lb/ft³ was obtained from International Paper. Each carton was foldedflat, and then fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder was equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades was adjusted to 0.10inch. The output from the shredder resembled confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inchand a thickness equivalent to that of the starting material (about 0.075inch).

The confetti-like material was fed to a Munson rotary knife cutter,Model SC30. Model SC30 is equipped with four rotary blades, four fixedblades, and a discharge screen having ⅛ inch openings. The gap betweenthe rotary and fixed blades was set to approximately 0.020 inch. Therotary knife cutter sheared the confetti-like pieces across theknife-edges, tearing the pieces apart and releasing a fibrous materialat a rate of about one pound per hour. The fibrous material had a BETsurface area of 0.9748 m2/g+/−0.0167 m²/g, a porosity of 89.0437 percentand a bulk density (@0.53 psia) of 0.1260 g/mL. An average length of thefibers was 1.141 mm and an average width of the fibers was 0.027 mm,giving an average LID of 42:1. A scanning electron micrograph of thefibrous material is shown in FIG. 26 at 25× magnification.

Example 2 Preparation of Fibrous Material from Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch and a thickness equivalent to that of the startingmaterial (about 0.075 inch). The confetti-like material was fed to aMunson rotary knife cutter, Model SC30. The discharge screen had ⅛ inchopenings. The gap between the rotary and fixed blades was set toapproximately 0.020 inch. The rotary knife cutter sheared theconfetti-like pieces, releasing a fibrous material at a rate of aboutone pound per hour. The fibrous material had a BET surface area of1.1316 m2/g+/−0.0103 m2/g, a porosity of 88.3285 percent and a bulkdensity (@0.53 psia) of 0.1497 g/mL. An average length of the fibers was1.063 mm and an average width of the fibers was 0.0245 mm, giving anaverage LID of 43:1. A scanning electron micrograph of the fibrousmaterial is shown in FIG. 27 at 25× magnification.

Example 3 Preparation of Twice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had 1/16 inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. The material resultingfrom the first shearing was fed back into the same setup described aboveand sheared again. The resulting fibrous material had a BET surface areaof 1.4408 m2/g+/−0.0156 m2/g, a porosity of 90.8998 percent and a bulkdensity (@0.53 psia) of 0.1298 g/mL. An average length of the fibers was0.891 mm and an average width of the fibers was 0.026 mm, giving anaverage LID of 34:1. A scanning electron micrograph of the fibrousmaterial is shown in FIG. 28 at 25× magnification.

Example 4 Preparation of Thrice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had ⅛ inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter sheared the confetti-like pieces across theknife-edges. The material resulting from the first shearing was fed backinto the same setup and the screen was replaced with a 1/16 inch screen.This material was sheared. The material resulting from the secondshearing was fed back into the same setup and the screen was replacedwith a 1/32 inch screen. This material was sheared. The resultingfibrous material had a BET surface area of 1.6897 m2/g+/−0.0155 m2/g, aporosity of 87.7163 percent and a bulk density (@0.53 psia) of 0.1448g/mL. An average length of the fibers was 0.824 mm and an average widthof the fibers was 0.0262 mm, giving an average LID of 32:1. A scanningelectron micrograph of the fibrous material is shown in FIG. 29 at 25×magnification.

Example 5 Preparation of Densified Fibrous Material from Bleached KraftBoard without Added Binder

Fibrous material was prepared according to Example 2. Approximately 1 lbof water was sprayed onto each 10 lb of fibrous material. The fibrousmaterial was densified using a California Pellet Mill 1100 operating at75° C. Pellets were obtained having a bulk density ranging from about 7lb/ft to about 15 lb/ft.

Example 6 Preparation of Densified Fibrous Material from Bleached KraftBoard with Binder

Fibrous material was prepared according to Example 2.

A 2 weight percent stock solution of POLYOX™ WSR N10 (polyethyleneoxide) was prepared in water.

Approximately 1 lb of the stock solution was sprayed onto each 10 lb offibrous material. The fibrous material was densified using a CaliforniaPellet Mill 1100 operating at 75° C. Pellets were obtained having a bulkdensity ranging from about 15 lb/ft to about 40 lb/ft.

Example 7 Reducing the Molecular Weight of Cellulose in Fibrous KraftPaper by Gamma Radiation with Minimum Oxidation

Fibrous material is prepared according to Example 4. The fibrous Kraftpaper is densified according to Example 5.

The densified pellets are placed in a glass ampoule having a maximumcapacity of 250 mL. The glass ampoule is evacuated under high vacuum(10⁻⁵ torr) for 30 minutes, and then back-filled with argon gas. Theampoule is sealed under argon. The pellets in the ampoule are irradiatedwith gamma radiation for about 3 hours at a dose rate of about 1 Mradper hour to provide an irradiated material in which the cellulose has alower molecular weight than the fibrous Kraft starting material.

Example 8 Reducing the Molecular Weight of Cellulose in Fibrous KraftPaper by Gamma Radiation with Maximum Oxidation

Fibrous material is prepared according to Example 4. The fibrous Kraftpaper is densified according to Example 5.

The densified pellets are placed in a glass ampoule having a maximumcapacity of 250 mL. The glass ampoule is sealed under an atmosphere ofair. The pellets in the ampoule are irradiated with gamma radiation forabout 3 hours at a dose rate of about 1 Mrad per hour to provide anirradiated material in which the cellulose has a lower molecular weightthan the fibrous Kraft starting material.

Example 9 Electron Beam Processing

Samples were treated with electron beam using a vaulted Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 1 describes the parameters used. Table 2 reports thenominal dose used for the Sample ID (in Mrad) and the corresponding dosedelivered to the sample (in kgy).

TABLE 1 Rhodotron ® TT 200 Parameters Beam Beam Produced: Acceleratedelectrons Beam energy: Norminal (fixed): 10 MeV (+0 keV-250 keV Energydispersion at 10 Mev: Full width half maximum (FWHM) 300 keV Beam powerat 10 MeV: Guaranteed Operating Range 1 to 80 kW Power ConsumptionStand-by condition (vacuum and  <15 kW cooling ON): At 50 kW beam power:<210 kW At 80 kW beam power: <260 kW RF System Frequency: 107.5 ± 1 MHzTetrode type: Thomson TH781 Scanning Horn Nominal Scanning Length(measured 120 cm at 25-35 em from window: Scanning Range: From 30% to100% of Nominal Scanning Length Nominal Scanning Frequency (at max. 100Hz ± 5% scanning length): Scanning Uniformity (across 90% of ±5% NominalScanning Length)

TABLE 2 Dosages Delivered to Samples Total Dosage (Mrad) (NumberAssociated with Sample ID Delivered Dose (kgy)¹ 1 9.9 3 29.0 5 50.4 769.2 10 100.0 15 150.3 20 198.3 30 330.9 50 529.0 70 695.9 100 993.6¹For example, 9.9 kgy was delivered in 11 seconds at a beam current of 5mA and a line speed of 12.9 feet/minute. Cool time between treatmentswas around 2 minutes.

Example 10 Methods of Determining Molecular Weight of Cellulosic andLignocellulosic Materials by Gel Permeation Chromatography

Cellulosic and lignocellulosic materials for analysis were treatedaccording to Example 4. Sample materials presented in the followingtables include Kraft paper (P), wheat straw (WS), alfalfa (A), cellulose(C), switchgrass (SG), grasses (G), and starch (ST), and sucrose (S).The number “132” of the Sample ID refers to the particle size of thematerial after shearing through a 1/32 inch screen. The number after thedash refers to the dosage of radiation (MRad) and “US” refers toultrasonic treatment. For example, a sample ID “P132-10” refers to Kraftpaper that has been sheared to a particle size of 132 mesh and has beenirradiated with 10 Mrad.

For samples that were irradiated withe-beam, the number following thedash refers to the amount of energy delivered to the sample. Forexample, a sample ID “P-100e” refers to Kraft paper that has beendelivered a dose of energy of about 100 MRad or about 1000 kgy (Table2).

TABLE 3 Peak Average Molecular weight of Irradiated Kraft Paper SampleAverage Mw +/− Source Sample ID Dosage (Mrad) Ultrasound Std. Dev. KraftP132 0 No 32853 ± 10006 Paper P132-10 10 ″  61398 ± 2468** P132-100 100″ 8444 ± 580  P132-181 181 ″ 6668 ± 77  P132-US 0 yes 3095 ± 1013 **Lowdoses of radiation appear to increase the molecular weight of somematerials ¹Dosage Rate = 1 MRadlhour ²Treatment for 30 minutes with 20kHz ultrasound using a 1000 W horn under re-circulating conditions withthe material dispersed in water.

TABLE 4 Peak Average Molecular Weight of Irradiated Kraft Paper withE-Beam Dosage Average MW ± Std. Sample Source Sample ID Mrad Dev. KraftPaper P-1e 1 63489 ± 595 P-Se 5 56587 ± 536 P-10e 10 53610 ± 327 P-30e30 38231 ± 124 P-70e 70 12011 ± 158 P-100e 100 9770 ± 2 

TABLE 5 Peak Average Molecular Weight of Gamma Irradiated MaterialsDosage¹ Average Mw +/− Sample ID Peak # Mrad Ultrasound² Std. Dev. WS1321 0 No 1407411 ± 175191 2 ″ ″ 39145 ± 3425 3 ″ ″ 2886 ± 177 WS132-10* 110 ″ 26040 ± 3240 WS132-100* 1 100 ″ 23620 ± 453  A132 1 0 ″ 1604886 ±151701 2 ″ ″ 37525 ± 3751 3 ″ ″ 2853 ± 490 A132-10* 1 10 ″ 50853 ± 16652 ″ ″ 2461 ± 17  A132-100* 1 100 ″ 38291 ± 2235 2 ″ ″ 2487 ± 15  SG132 10 ″ 1557360 ± 83693  2 ″ ″ 42594 ± 4414 3 ″ ″ 3268 ± 249 SG132-10* 1 10″ 60888 ± 9131 SG132-100* 1 100 ″ 22345 ± 3797 SG132-10-US 1 10 Yes 86086 ± 43518 2 ″ ″ 2247 ± 468 SG132-100-US 1 100 ″  4696 ± 1465 *Peakscoalesce after treatment **Low doses of radiation appear to increase themolecular weight of some materials ¹Dosage Rate = 1 MRadlhour ²Treatmentfor 30 minutes with 20 kHz ultrasound using a 1000 W horn underre-circulating conditions with the material dispersed in water.

TABLE 6 Peak Average Molecular Weight of Irradiated Material with E-BeamAverage MW ± Std. Sample ID Peak # Dosage Dev. A-1e 1 1 1004783 ± 975182 34499 ± 482 3 2235 ± 1  A-5e 1 5 38245 ± 346 2 2286 ± 35 A-10e 1 1044326 ± 33  2 2333 ± 18 A-30e 1 30 47366 ± 583 2 2377 ± 7  A-50e 1 5032761 ± 168 2 2435 ± 6  G-1e 1 1  447362 ± 38817 2 32165 ± 779 3 3004 ±25 G-5e 1 5  62167 ± 6418 2 2444 ± 33 G-10e 1 10  72636 ± 4075 2 3065 ±34 G-30e 1 30 17159 ± 390 G-50e 1 50 18960 ± 142 ST 1 0 923336 ± 1883 2150265 ± 4033 ST-1e 1 1 846081 ± 5180 2 131222 ± 1687 ST-5e 1 5  90664 ±1370 ST-10e 1 10 98050 ± 255 ST-30e 1 30 41884 ± 223 ST-70e 1 70 9699 ±31 ST-100e 1 100 8705 ± 38

Gel Permeation Chromatography (GPC) is used to determine the molecularweight distribution of polymers. During GPC analysis, a solution of thepolymer sample is passed through a column packed with a porous geltrapping small molecules. The sample is separated based on molecularsize with larger molecules eluting sooner than smaller molecules. Theretention time of each component is most often detected by refractiveindex (RI), evaporative light scattering (ELS), or ultraviolet (UV) andcompared to a calibration curve. The resulting data is then used tocalculate the molecular weight distribution for the sample.

A distribution of molecular weights rather than a unique molecularweight is used to characterize synthetic polymers. To characterize thisdistribution, statistical averages are utilized. The most common ofthese averages are the “number average molecular weight” (Mn) and the“weight average molecular weight” (Mw).

Mn is similar to the standard arithmetic mean associated with a group ofnumbers. When applied to polymers, Mn refers to the average molecularweight of the molecules in the polymer. Mn is calculated affording thesame amount of significance to each molecule regardless of itsindividual molecular weight. The average M_(n) is calculated by thefollowing formula where N is the number of molecules with a molar massequal to M_(i).

${\overset{\_}{M}}_{n} = \frac{\sum_{i}{N_{i}M_{i}}}{\sum_{i}N_{i}}$

M_(w) is another statistical descriptor of the molecular weightdistribution that places a greater emphasis on larger molecules thansmaller molecules in the distribution. The formula below shows thestatistical calculation of the weight average molecular weight.

${\overset{\_}{M}}_{n} = \frac{\sum_{i}{N_{i}M_{i}^{2}}}{\sum_{i}{N_{i}M_{i}}}$

The polydispersity index or PI is defined as the ratio of Mw/Mn. Thelarger the PI, the broader or more disperse the distribution. The lowestvalue that a PI can be is 1. This represents a monodisperse sample; thatis, a polymer with all of the molecules in the distribution being thesame molecular weight.

The peak molecular weight value (Mp) is another descriptor defined asthe mode of the molecular weight distribution. It signifies themolecular weight that is most abundant in the distribution. This valuealso gives insight to the molecular weight distribution.

Most GPC measurements are made relative to a different polymer standard.The accuracy of the results depends on how closely the characteristicsof the polymer being analyzed match those of the standard used. Theexpected error in reproducibility between different series ofdeterminations, calibrated separately, is ca. 5-10% and ischaracteristic to the limited precision of GPC determinations.Therefore, GPC results are most useful when a comparison between themolecular weight distributions of different samples is made during thesame series of determinations.

The lignocellulosic samples required sample preparation prior to GPCanalysis. First, a saturated solution (8.4% by weight) of lithiumchloride (LiCl) was prepared in dimethyl acetamide (DMAc). Approximately100 mg of the sample was added to approximately 10 g of a freshlyprepared saturated LiCl/DMAc solution, and the mixture was heated toapproximately 150° C.-170° C. with stirring for 1 hour. The resultingsolutions were generally light- to dark-yellow in color. The temperatureof the solutions were decreased to approximately 100° C. and heated foran additional 2 hours. The temperatures of the solutions were thendecreased to approximately 50° C. and the sample solution was heated forapproximately 48 to 60 hours. Of note, samples irradiated at 100 MRadwere more easily solubilized as compared to their untreated counterpart.Additionally, the sheared samples (denoted by the number 132) hadslightly lower average molecular weights as compared with uncut samples.

The resulting sample solutions were diluted 1:1 using DMAc as solventand were filtered through a 0.45 μm PTFE filter. The filtered samplesolutions were then analyzed by GPC using the parameters described inTable 7. The peak average molecular weights (Mp) of the samples, asdetermined by Gel Permeation Chromatography (GPC), are summarized inTables 3-6. Each sample was prepared in duplicate and each preparationof the sample was analyzed in duplicate (two injections) for a total offour injections per sample. The EasiCal® polystyrene standards PS1A andPS1B were used to generate a calibration curve for the molecular weightscale from about 580 to 7,500,00 Daltons.

TABLE 7 GPC Analysis Conditions Instrument: Waters Alliance GPC 2000Colums (3): Pigel 10 μ Mixed-B S/N's: 10M-MB-148-83; 10M-MB- 148-84;10M-174-129 Mobile Phase (Solvent) 0.5% LiCl in DMAc (1.0 mL/min.)Column/Detector Temperature 70° C. Injector Temperature: 70° C. SampleLoop Size: 323.5 μL

Example 11 Time-Of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS)Surface Analysis

Time-of-Flight Secondary Ion Mass Spectroscopy (ToP-SIMS) is asurface-sensitive spectroscopy that uses a pulsed ion beam (Cs ormicrofocused Ga) to remove molecules from the very outermost surface ofthe sample. The particles are removed from atomic monolayers on thesurface (secondary ions). These particles are then accelerated into a“flight tube” and their mass is determined by measuring the exact timeat which they reach the detector (i.e. time-of-flight). ToP-SIMSprovides detailed elemental and molecular information about the surface,thin layers, interfaces of the sample, and gives a fullthree-dimensional analysis. The use is widespread, includingsemiconductors, polymers, paint, coatings, glass, paper, metals,ceramics, biomaterials, pharmaceuticals and organic tissue. SinceToP-SIMS is a survey technique, all the elements in the periodic table,including H, are detected. ToF-SIMS data is presented in Tables 8-11.Parameters used are reported in Table 12.

TABLE 8 Normalized Mean Intensities of Various Positive Ions of Interest(Normalized relative to total ion counts × 10000) P132 P132-10 P132-100m/z Species Mean σ Mean σ Mean σ 23 Na 257 28 276 54 193 36 27 Al 647 43821 399 297 44 28 Si 76 45.9 197 89 81.7 10.7 15 CH₃ 77.9 7.8 161 26 13312 27 C₂H₃ 448 28 720 65 718 82 39 C₃H₃ 333 10 463 37 474 26 41 C₃H₅ 70319 820 127 900 63 43 C₃H₇ 657 11 757 162 924 118 115 C₉H₇ 73 13.4 40.34.5 42.5 15.7 128 C₁₀H₈ 55.5 11.6 26.8 4.8 27.7 6.9 73 C₃H₉Si* 181 7765.1 18.4 81.7 7.5 147 C₅H₁₅OSi₂* 72.2 33.1 24.9 10.9 38.5 4 207C₅H₁₅O₃Si₃* 17.2 7.8 6.26 3.05 7.49 1.77 647 C₄₂H₅₄P0₃ 3.63 1.05 1.431.41 10.7 7.2

TABLE 9 Normalized Mean Intensities of Various Negative Ions of Interest(Normalized relative to total ion counts × 10000) P132 P132-10 P132-100m/z Species Mean σ Mean σ Mean σ 19 F 15.3 2.1 42.4 37.8 19.2 1.9 35 Cl63.8 2.8 107 33 74.1 5.5 13 CH 1900 91 1970 26 1500 6 25 C₂H 247 127 22099 540 7 26 CN 18.1 2.1 48.6 30.8 43.9 1.4 42 CNO 1.16 0.71 0.743 0.71110.8 0.9 46 NO₂ 1.87 0.38 1.66 1.65 12.8 1.8

TABLE 10 Normalized Mean Intensities of Various Positive Ions ofInterest (Normalized relative to total ion counts × 10000) P-1e P-5eP-10e P-30e P-70e P-100e m/z Species Mean σ Mean σ Mean σ Mean σ Mean σMean σ 23 Na 232 56 370 37 241 44 518 57 350 27 542 104 27 Al 549 194677 86 752 371 761 158 516 159 622 166 28 Si 87.3 11.3 134 24 159 100158 32 93.7 17.1 124 11 15 CH₃ 114 23 92.9 3.9 128 18 110 16 147 16 1415 27 C₂H₃ 501 205 551 59 645 165 597 152 707 94 600 55 39 C₃H₃ 375 80288 8 379 82 321 57 435 61 417 32 41 C₃H₅ 716 123 610 24 727 182 607 93799 112 707 84 43 C₃H₇ 717 121 628 52 653 172 660 89 861 113 743 73 115C₉H₇ 49.9 14.6 43.8 2.6 42.2 7.9 41.4 10.1 27.7 8 32.4 10.5 128 C₁₀H₈38.8 13.1 39.2 1.9 35.2 11.8 31.9 7.8 21.2 6.1 24.2 6.8 73 C₃H₉Si* 92.53.0 80.6 2.9 72.3 7.7 75.3 11.4 63 3.4 55.8 2.1 147 C₅H₁₅OSi₂* 27.2 3.917.3 1.2 20.4 4.3 16.1 1.9 21.7 3.1 16.3 1.7 207 C₅H₁₅O₃Si₃* 6.05 0.743.71 0.18 4.51 0.55 3.54 0.37 5.31 0.59 4.08 0.28 647 C₄₂H₅₄P0₃ 1.611.65 1.09 1.30 0.325 0.307 nd ~ 0.868 1.31 0.306 0.334

TABLE 11 Normalized Mean Intensities of Various Negative Ions ofInterest (Normalized relative to total ion counts × 10000) P-1e P-5eP-10e P-30e P-70e P-100e m/z Species Mean σ Mean σ Mean σ Mean σ Mean σMean σ 13 CH 1950 72 1700 65 1870 91 1880 35 2000 46 2120 102 25 C₂H 15447 98.8 36.3 157 4 230 17 239 22 224 19 19 F 25.4 1 24.3 1.4 74.3 18.640.6 14.9 25.6 1.9 21.5 2 35 Cl 39.2 13.5 38.7 3.5 46.7 5.4 67.6 6.245.1 2.9 32.9 10.2 26 CN 71.9 18.9 6.23 2.61 28.1 10.1 34.2 29.2 57.328.9 112 60 42 CNO 0.572 0.183 0.313 0.077 0.62 0.199 1.29 0.2 1.37 0.551.38 0.28 46 NO₂ 0.331 0.057 0.596 0.255 0.668 0.149 1.44 0.19 1.92 0.290.549

TABLE 12 Tof-SIMS Parameters Instrument Conditions: Instrument: PHITRIFT II Primary Ion Source: ⁶⁹Ga Primary Ion Beam Potential: 12 kV +ions 18 kV − ions Primary Ion Current (DC): 2 na for P#E samples 600 pAfor P132 samples Energy Filter/CD: Out/Out Masses Blanked: None ChargeCompensation: On

Tof-SIMS uses a focused, pulsed particle beam (typically Cs or Ga) todislodge chemical species on a materials surface. Particles producedcloser to the site of impact tend to be dissociated ions (positive ornegative). Secondary particles generated farther from the impact sitetend to be molecular compounds, typically fragments of much largerorganic macromolecules. The particles are then accelerated into a flightpath on their way towards a detector. Because it is possible to measurethe “time-of-flight” of the particles from the time of impact todetector on a scale of nano-seconds, it is possible to produce a massresolution as fine as O.OOX atomic mass units (i.e. one part in athousand of the mass of a proton). Under typical operating conditions,the results of ToF-SIMS analysis include: a mass spectrum that surveysall atomic masses over a range of 0-10,000 amu, the rastered beamproduces maps of any mass of interest on a sub-micron scale, and depthprofiles are produced by removal of surface layers by sputtering underthe ion beam. Negative ion analysis showed that the polymer hadincreasing amounts of CNO, CN, and NO₂ groups.

Example 12 X-Ray Photoelectron Spectroscopy (XPS) Electron Spectroscopyfor Chemical Analysis (ESCA)

X-Ray Photoelectron Spectroscopy (XPS) (sometimes called “ESCA”)measures the chemical composition of the top five nanometers of surface;XPS uses photo-ionization energy and energy-dispersive analysis of theemitted photoelectrons to study the composition and electronic state ofthe surface region of a sample. X-ray Photoelectron spectroscopy isbased upon a single photon in/electron out. Soft x-rays stimulate theejection of photoelectrons whose kinetic energy is measured by anelectrostatic electron energy analyzer. Small changes to the energy arecaused by chemically-shifted valence states of the atoms from which theelectrons are ejected; thus, the measurement provides chemicalinformation about the sample surface.

TABLE 13 Atomic Concentrations (in %)^(a,b) Atom Sample ID C O Al SiP132 (Area 1) 57.3 39.8 1.5 1.5 P132 (Area 2) 57.1 39.8 1.6 1.5 A132-10(Area 1) 63.2 33.5 1.7 1.6 A132-10 (Area 2) 65.6 31.1 1.7 1.7 A132-100(Area 1) 61.2 36.7 0.9 1.0 A132-100 (Area 2) 61 36.9 0.8 1.3^(a)Normalized to 100% of the elements detected. XPS does not detect Hor He.

TABLE 14 Carbon Chemical State (in % C) C—C, C—H C—O C═O O—C═O P132(Area 1) 22 49 21 7 P132 (Area 2) 25 49 20 6 P132-10 (Area 1) 34 42 15 9P132-10 (Area 2) 43 38 14 5 P132-100 (Area 1) 27 45 15 9 P132-100 (Area2) 25 44 23 9

TABLE 15 Atomic Concentrations (in %)^(a,b) Atom Sample ID C O Al Si NaP-1e (Area 1) 59.8 37.9 1.4 0.9 ~ P-1e (Area 2) 58.5 38.7 1.5 1.3 ~ P-Se(Area 1) 58.1 39.7 1.4 0.8 ~ P-Se (Area 2) 58.0 39.7 1.5 0.8 ~ P-1Oe(Area 1) 61.6 36.7 1.1 0.7 ~ P-1Oe (Area 2) 58.8 38.6 1.5 1.1 ~ P-SOe(Area 1) 59.9 37.9 1.4 0.8 <0.1 P-SOe (Area 2) 59.4 38.3 1.4 0.9 <0.1P-70e (Area 1) 61.3 36.9 1.2 0.6 <0.1 P-70e (Area 2) 61.2 36.8 1.4 0.7<0.1 P-100e (Area 1) 61.1 37.0 1.2 0.7 <0.1 P-100e (Area 2) 60.5 37.21.4 0.9 <0.1 ^(a)Normalized to 100% of the elements detected. XPS doesnot detect H or He. ^(b)A less than symbol “<” indicates accuratequantification cannot be made due to weak signal intensity.

TABLE 16 Carbon Chemical State Table (in % C) Sample ID C—C, C—H C—O C═O0-C═O P-1e (Area 1) 29 46 20 5 P-1e (Area 2) 27 49 19 5 P-Se (Area 1) 2553 18 5 P-Se (Area 2) 28 52 17 4 P-10e (Area 1) 33 47 16 5 P-10e (Area2) 28 51 16 5 P-S0e (Area 1) 29 45 20 6 P-S0e (Area 1) 28 50 16 5 P-70e(Area 1) 32 45 16 6 P-70e (Area 2) 35 43 16 6 P-100e (Area 1) 32 42 19 7P-100e (Area 2) 30 47 16 7

TABLE 17 Analytical Parameters Instrument: PHI Quantum 2000 X-raysource: Monochromated Alka 1486.6 eV Acceptance Angle: ±23° Take-offangle:   45° Analysis area: 1400 × 300 IJm Charge Correction: C1s 284.8eV

XPS spectra are obtained by irradiating a material with a beam ofaluminum or magnesium X-rays while simultaneously measuring the kineticenergy (KE) and number of electrons that escape from the top 1 to 10 nmof the material being analyzed (see analytical parameters, Table 17).The XPS technique is highly surface specific due to the short range ofthe photoelectrons that are excited from the solid. The energy of thephotoelectrons leaving the sample is determined using a ConcentricHemispherical Analyzer (CHA) and this gives a spectrum with a series ofphotoelectron peaks. The binding energy of the peaks is characteristicof each element. The peak areas can be used (with appropriatesensitivity factors) to determine the composition of the materialssurface. The shape of each peak and the binding energy can be slightlyaltered by the chemical state of the emitting atom. Hence XPS canprovide chemical bonding information as well. XPS is not sensitive tohydrogen or helium, but can detect all other elements. XPS requiresultra-high vacuum (UHV) conditions and is commonly used for the surfaceanalysis of polymers, coatings, catalysts, composites, fibers, ceramics,pharmaceutical/medical materials, and materials of biological origin.XPS data is reported in Tables 13-16.

Example 13 Raman Analysis

Raman spectra were acquired from the surface of fibers from samples:P132, P132-100, P-Ie, and P-100e. The measurements were performed usinga “LabRam” J-Y Spectrometer. A HeNe laser (632.8 nm wavelength) and 600grimm grating were used for the measurements. The measurements wereperformed confocally using backscattering geometry (180°) under anOlympus BX40 microscope. The samples had a Raman spectrum typical ofcellulose.

Example 14 Scanning Probe Microscopy (SPM) Surface Analysis Using anAtomic Force Microscope (AFM)

The purpose of this analysis was to obtain Atomic Force Microscope (AFM)images of the samples in Tables 18 and 19 to measure surface roughness.

Scanning probe microscopy (SPM) is a branch of microscopy that formsimages of surfaces using a physical probe that scans the specimen. Animage of the surface is obtained by mechanically moving the probe in araster scan of the specimen, line by line, and recording theprobe-surface interaction as a function of position. The atomic forcemicroscope (AFM) or scanning force microscope (SFM) is a veryhigh-resolution type of scanning probe microscope, with demonstratedresolution of fractions of a nanometer, more than 1000 times better thanthe optical diffraction limit. The probe (or the sample under astationary probe) generally is moved by a piezoelectric tube. Suchscanners are designed to be moved precisely in any of the threeperpendicular axes (x,y,z). By following a raster pattern, the sensordata forms an image of the probe-surface interaction. Feedback from thesensor is used to maintain the probe at a constant force or distancefrom the object surface. For atomic force microscopy, the sensor is aposition-sensitive photodetector that records the angle of reflectionfrom a laser beam focused on the top of the cantilever.

TABLE 18 Roughness Results for Gamma-Irradiated Samples Sample ID RMS(A) R_(max) (Å) P132 927.2 716.3 8347.6 P132-10 825.7 576.8 11500P132-100 1008 813.5 7250.7

TABLE 19 Roughness Results for Samples Irradiated with E-Beam Sample IDRMS (A) Ra (A) Rmax (A) P-1e 1441.2 1147.1 8955.4 P-Se 917.3 727.56753.4 P-10e 805.6 612.1 7906.5 P-30e 919.2 733.7 6900 P-70e 505.8 388.15974.2 P-100e 458.2 367.9 3196.9

AFM images were collected using a NanoScope III Dimension 5000 (DigitalInstruments, Santa Barbara, Calif., USA). The instrument was calibratedagainst a NIST traceable standard with an accuracy better than 2%.NanoProbe silicon tips were used. Image processing procedures involvingauto-flattening, plane fitting or convolution were employed.

One 5 μm×5 μm area was imaged at a random location on top of a singlefiber. Perspective (3-D) views of these surfaces are included withvertical exaggerations noted on the plots (FIGS. 29A-29F). The roughnessanalyses were performed and are expressed in: (1) Root-Mean-SquareRoughness, RMS; (2) Mean Roughness, Ra; and (3) Maximum Height(Peak-to-Valley), Rmax. Results are summarized in Tables 18 and 19.

Example 15 Determining Crystallinity of Irradiated Materials by X-RayDiffraction

X-ray diffraction (XRD) is a method by which a crystalline sample isirradiated with monoenergetic x-rays. The interaction of the latticestructure of the sample with these x-rays is recorded and providesinformation about the crystalline structure being irradiated. Theresulting characteristic “fingerprint” allows for the identification ofthe crystalline compounds present in the sample. Using a whole-patternfitting analysis (the Rietvelt Refinement), it is possible to performquantitative analyses on samples containing more than one crystallinecompound.

TABLE 20 XRD Data Including Domain Size and % Crystallinity Domain SizeSample ID (Å) % Crystallinity P132 55 55 P132-10 46 58 P132-100 50 55P132-181 48 52 P132-US 26 40 A132 28 42 A132-10 26 40 A132-100 28 35WS132 30 36 WS132-10 27 37 WS132-100 30 41 SG132 29 40 SG132-10 28 38SG132-100 28 37 SG132-10-US 25 42 SG132-100-US 21 34

Each sample was placed on a zero background holder and placed in aPhillips PW1800 diffractometer using Cu radiation. Scans were then runover the range of 5° to 50° with a step size of 0.05° and a countingtime of 2 hours each.

Once the diffraction patterns were obtained, the phases were identifiedwith the aid of the Powder Diffraction File published by theInternational Centre for Diffraction Data. In all samples thecrystalline phase identified was cellulose—Ia, which has a triclinicstructure.

The distinguishing feature among the 20 samples is the peak breadth,which is related to the crystallite domain size. The experimental peakbreadth was used to compute the domain size and percent crystallinity,which are reported in Table 4.

Percent crystallinity (Xc %) is measured as a ratio of the crystallinearea to the total area under the x-ray diffraction peaks,

${X_{c}\%} = {\frac{A_{c}}{\left\{ {A_{0} + A_{C}} \right\}} \times 100\%}$where,

-   -   A_(c)=Area of crystalline phase    -   A_(a)=Area of amorphous phase    -   X_(c)=Percent of crystallinity

To determine the percent crystallinity for each sample it was necessaryto first extract the amount of the amorphous phase. This is done byestimating the area of each diffraction pattern that can be attributedto the crystalline phase (represented by the sharper peaks) and thenon-crystalline phase (represented by the broad humps beneath thepattern and centered at 22° and 38°).

A systematic process was used to minimize error in these calculationsdue to broad crystalline peaks as well as high background intensity.First, a linear background was applied and then removed. Second, twoGaussian peaks centered at 22° and 38° with widths of 10-12° each werefitted to the humps beneath the crystalline peaks. Third, the areabeneath the two broad Gaussian peaks and the rest of the pattern weredetermined. Finally, percent crystallinity was calculated by dividingthe area beneath the crystalline peak by the total intensity (afterbackground subtraction). Domain size and % crystallinity of the samplesas determined by X-ray diffraction (XRD) are presented in Table 20.

Example 16 Porosimetry Analysis of Irradiated Materials

Mercury pore size and pore volume analysis (Table 21) is based onforcing mercury (a non-wetting liquid) into a porous structure undertightly controlled pressures. Since mercury does not wet most substancesand will not spontaneously penetrate pores by capillary action, it mustbe forced into the voids of the sample by applying external pressure.The pressure required to fill the voids is inversely proportional to thesize of the pores. Only a small amount of force or pressure is requiredto fill large voids, whereas much greater pressure is required to fillvoids of very small pores.

TABLE 21 Pore Size and Volume Distribution by Mercury Porosimetry MedianMedian Average Bulk Total Pore Pore Pore Density Apparent IntrusionDiameter Diameter Diameter @0.50 (skeletal) Sample Volume Total Pore(Volume) (Area) (4 V/A) psia Density Porosity ID (mL/g) m²/g) (μm) (μm)(μm) (g/mL) (g/mL) (%) P132 6.0594 1.228 36.2250 13.7278 19.7415 0.14481.1785 87.7163 P132-10 5.5436 1.211 46.3463 4.5646 18.3106 0.1614 1.535589.4875 P132-100 5.3985 0.998 34.5235 18.2005 21.6422 0.1612 1.241387.0151 P132-181 3.2866 0.868 25.3448 12.2410 15.1509 0.2497 1.391682.0577 P132-US 6.0005 14.787 98.3459 0.0055 1.6231 0.1404 0.889484.2199 A132 2.0037 11.759 64.6308 0.0113 0.6816 0.3683 1.4058 73.7990A132-10 1.9514 10.326 53.2706 0.0105 0.7560 0.3768 1.4231 73.5241A132-100 1.9394 10.205 43.8966 0.0118 0.7602 0.3760 1.3889 72.9264 SG1322.5267 8.265 57.6958 0.0141 1.2229 0.3119 1.4708 78.7961 SG132-10 2.14148.643 26.4666 0.0103 0.9910 0.3457 1.3315 74.0340 SG132-100 2.514210.766 32.7118 0.0098 0.9342 0.3077 1.3590 77.3593 SG132-10-US 4.40431.722 71.5734 1.1016 10.2319 0.1930 1.2883 85.0169 SG132-100-US 4.96657.358 24.8462 0.0089 2.6998 0.1695 1.0731 84.2010 WS132 2.9920 5.44776.3675 0.0516 2.1971 0.2773 1.6279 82.9664 WS132-10 3.1138 2.90157.4727 0.3630 4.2940 0.2763 1.9808 86.0484 WS132-100 3.2077 3.11452.3284 0.2876 4.1199 0.2599 1.5611 83.3538 A-1e 1.9535 3.698 25.34110.0810 2.1130 0.3896 1.6299 76.0992 A-5e 1.9697 6.503 29.5954 0.03361.2117 0.3748 1.4317 73.8225 A-10e 2.0897 12.030 45.5493 0.0101 0.69480.3587 1.4321 74.9545 A-50e 2.1141 7.291 37.0760 0.0304 1.1599 0.35771.4677 75.6264 G-le 2.4382 7.582 58.5521 0.0201 1.2863 0.3144 1.347276.6610 G-5e 2.4268 6.436 44.4848 0.0225 1.5082 0.3172 1.3782 76.9831G-10e 2.6708 6.865 62.8605 0.0404 1.5562 0.2960 1.4140 79.0638 G-50e2.8197 6.798 56.5048 0.0315 1.6591 0.2794 1.3179 78.7959 P-1e 7.76921.052 49.8844 22.9315 29.5348 0.1188 1.5443 92.3065 P-Se 7.1261 1.21246.6400 12.3252 23.5166 0.1268 1.3160 90.3644 P-10e 6.6096 1.113 41.425217.4375 23.7513 0.1374 1.4906 90.7850 P-50e 6.5911 1.156 40.7837 15.982322.7974 0.1362 1.3302 89.7616 P-100e 5.3507 1.195 35.3622 10.740017.9063 0.1648 1.3948 88.1840 S 0.4362 0.030 102.8411 42.5047 57.82080.9334 1.5745 40.7160 S-1e 0.3900 0.632 90.6808 0.0041 2.4680 0.97721.5790 38.1140 S-5e 0.3914 0.337 97.1991 0.0070 4.6406 0.9858 1.605238.5847 S-10e 0.4179 0.349 113.4360 0.0042 4.7873 0.9469 1.5669 39.5678S-30e 0.4616 5.329 102.0559 0.0042 0.3464 0.9065 1.5585 41.8388 S-50e0.5217 7.162 137.2124 0.0051 0.2914 0.8521 1.5342 44.4582 S-100e 0.881715.217 76.4577 0.0053 0.2318 0.6478 1.5105 57.1131 St 0.6593 17.6314.2402 0.0053 0.1496 0.7757 1.5877 51.1438 St-le 0.6720 18.078 4.33600.0052 0.1487 0.7651 1.5750 51.4206 St-5e 0.6334 19.495 4.2848 0.00510.1300 0.7794 1.5395 49.3706 St-10e 0.6208 16.980 4.3362 0.0056 0.14620.7952 1.5703 49.3630 St-30e 0.6892 18.066 4.4152 0.0050 0.1526 0.74751.5417 51.5465 S-50e 0.6662 18.338 4.3759 0.0054 0.1453 0.7637 1.554850.8778 S-100e 0.6471 23.154 5.4032 0.0048 0.1118 0.7229 1.3582 46.7761

The AutoPore 9520 can attain a maximum pressure of 414 MPa or 60,000psia. There are four low pressure stations for sample preparation andcollection of macropore data from 0.2 psia to 50 psia. There are twohigh pressure chambers, which collect data from 25 psia to 60,000 psia.The sample is placed in a bowl-like apparatus called a penetrometer,which is bonded to a glass capillary stem with a metal coating. Asmercury invades the voids in and around the sample, it moves down thecapillary stem. The loss of mercury from the capillary stem results in achange in the electrical capacitance. The change in capacitance duringthe experiment is converted to volume of mercury by knowing the stemvolume of the penetrometer in use. A variety of penetrometers withdifferent bowl (sample) sizes and capillaries are available toaccommodate most sample sizes and configurations. Table 22 below definessome of the key parameters calculated for each sample.

TABLE 22 Definition of Parameters Parameter Description Total IntrusionThe total volume of mercury intruded during an Volume: experiment. Thiscan include interstitial filling between small particles, porosity ofsample, and compression volume of sample. Total Pore Area: The totalintrusion volume converted to an area assuming cylindrical shaped pores.Median Pore The size at the 50^(th) percentile on the cumulativeDiameter volume graph. (volume): Median Pore The size at the 50^(th)percentile on the cumulative area Diameter (area): graph. Average PoreThe total pore volume divided by the total pore area Diameter: (4VIA).Bulk Density: The mass of the sample divided by the bulk volume. Bulkvolume is determined at the filling pressure, typically 0.5 psia.Apparent Density: The mass of sample divided by the volume of samplemeasured at highest pressure, typically 60,000 psia. Porosity: (BulkDensity/Apparent Density) × 100%

Example 17 Particle Size Analysis of Irradiated Materials

The technique of particle sizing by static light scattering is based onMie theory (which also encompasses Fraunhofer theory). Mie theorypredicts the intensity vs. angle relationship as a function of the sizefor spherical scattering particles provided that other system variablesare known and held constant. These variables are the wavelength ofincident light and the relative refractive index of the sample material.Application of Mie theory provides the detailed particle sizeinformation. Table 23 summarizes particle size using median diameter,mean diameter, and modal diameter as parameters.

TABLE 23 Particle Size by Laser Light Scattering (Dry Sample Dispersion)Median Mean Diameter Sample ID Diameter (tm) (tm) Modal Diameter (tm)A132 380.695 418.778 442.258 A132-10 321.742 366.231 410.156 A132-100301.786 348.633 444.169 SG132 369.400 411.790 455.508 SG132-10 278.793325.497 426.717 SG132-100 242.757 298.686 390.097 WS132 407.335 445.618467.978 WS132-10 194.237 226.604 297.941 WS132-100 201.975 236.037307.304

Particle size was determined by Laser Light Scattering (Dry SampleDispersion) using a Malvern Mastersizer 2000 using the followingconditions:

-   -   Feed Rate: 35%    -   Disperser Pressure: 4 Bar    -   Optical Model: (2.610, 1.000i), 1.000

An appropriate amount of sample was introduced onto a vibratory tray.The feed rate and air pressure were adjusted to ensure that theparticles were properly dispersed. The key component is selecting an airpressure that will break up agglomerations, but does not compromise thesample integrity. The amount of sample needed varies depending on thesize of the particles. In general, samples with fine particles requireless material than samples with coarse particles.

Example 18 Surface Area Analysis of Irradiated Materials

Surface area of each sample was analyzed using a Micromeritics ASAP 2420Accelerated Surface Area and Porosimetry System. The samples wereprepared by first degassing for 16 hours at 40° C. Next, free space(both warm and cold) with helium is calculated and then the sample tubeis evacuated again to remove the helium. Data collection begins afterthis second evacuation and consists of defining target pressures, whichcontrol how much gas is dosed onto the sample. At each target pressure,the quantity of gas adsorbed and the actual pressure are determined andrecorded. The pressure inside the sample tube is measured with apressure transducer. Additional doses of gas will continue until thetarget pressure is achieved and allowed to equilibrate. The quantity ofgas adsorbed is determined by summing multiple doses onto the sample.The pressure and quantity define a gas adsorption isotherm and are usedto calculate a number of parameters, including BET surface area (Table24).

TABLE 24 Summary of Surface Area by Gas Adsorption BET Sample ID Singlepoint surface area (m²/g) Surface Area (m²/g) P132 @P/Po = 0.2503877711.5253 1.6897 P132-10 @P/Po = 0.239496722 1.0212 1.2782 P132-100 @P/Po =0.240538100 1.0338 1.2622 P132-181 @P/Po = 0.239166091 0.5102 0.6448P132-US @P/Po = 0.217359072 1.0983 1.6793 A132 @P/Po = 0.2400406100.5400 0.7614 A132-10 @P/Po = 0.211218936 0.5383 0.7212 A132-100 @P/Po =0.238791097 0.4258 0.5538 SG132 @P/Po = 0.237989353 0.6359 0.8350SG132-10 @P/Po = 0.238576905 0.6794 0.8689 SG132-100 @P/Po = 0.2419603610.5518 0.7034 SG132-10-US @P/Po = 0.225692889 0.5693 0.7510 SG132-100-US@P/Po = 0.225935246 1.0983 1.4963 G-10-US 0.751 G100-US 1.496 G132-US1.679 WS132 @P/Po = 0.237823664 0.6582 0.8663 WS132-10 @P/Po =0.238612476 0.6191 0.7912 WS132-100 @P/Po = 0.238398832 0.6255 0.8143

The BET model for isotherms is a widely used theory for calculating thespecific surface area. The analysis involves determining the monolayercapacity of the sample surface by calculating the amount required tocover the entire surface with a single densely packed layer of krypton.The monolayer capacity is multiplied by the cross sectional area of amolecule of probe gas to determine the total surface area. Specificsurface area is the surface area of the sample aliquot divided by themass of the sample.\

Example 19 Fiber Length Determination of Irradiated Materials

Fiber length distribution testing was performed in triplicate on thesamples submitted using the Techpap MorFi LBO1 system. The average fiberlength and width are reported in Table 25.

TABLE 25 Summary of Lignocellulosic Fiber Length and Width DataStatistically Corrected Average Average Arithmetic Length Length WidthAverage Weighted in Weighted (micrometers) Sample ID (mm) Length (mm) inLength (mm) (μm) P132-10 0.484 0.615 0.773 24.7 P132-100 0.369 0.4230.496 23.8 P132-181 0.312 0.342 0.392 24.4 A132-10 0.382 0.423 0.65043.2 A132-100 0.362 0.435 0.592 29.9 SG132-10 0.328 0.363 0.521 44.0SG132-100 0.325 0.351 0.466 43.8 WS132-10 0.353 0.381 0.565 44.7WS132-100 0.354 0.371 0.536 45.4

Example 20 Ultrasonic Treatment of Irradiated and Un-IrradiatedSwitchgrass

Switchgrass was sheared according to Example 4. The switchgrass wastreated by ultrasound alone or irradiation with 10 Mrad or 100 Mrad ofgamma rays, and then sonicated. The resulting materials correspond toG132-BR (un-irradiated), G132-10-BR (10 Mrad and sonication) andG132-100-BR (100 Mrad and sonication), as presented in Table 1.Sonication was performed on each sample for 30 minutes using 20 kHzultrasound from a 1000 W horn under re-circulating conditions. Eachsample was dispersed in water at a concentration of about 0.10 g/mL.

FIGS. 30 and 31 show the apparatus used for sonication. Apparatus 500includes a converter 502 connected to booster 504 communicating with ahorn 506 fabricated from titanium or an alloy of titanium. The horn,which has a seal 510 made from VITON® about its perimeter on itsprocessing side, forms a liquid tight seal with a processing cell 508.The processing side of the horn is immersed in a liquid, such as water,that has dispersed therein the sample to be sonicated. Pressure in thecell is monitored with a pressure gauge 512. In operation, each sampleis moved by pump 517 from tank 516 through the processing cell and issonicated. After, sonication, the sample is captured in tank 520. Theprocess can be reversed in that the contents of tank 520 can be sentthrough the processing cell and captured in tank 516. This process canbe repeated a number of times until a desired level of processing isdelivered to the sample.

Example 21 Scanning Electron Micrographs of Un-Irradiated Switchgrass inComparison to Irradiated and Irradiated and Sonicated Switchgrass

Switchgrass samples for the scanning electron micrographs were appliedto carbon tape and gold sputter coated (70 seconds). Images were takenwith a JEOL 6500 field emission scanning electron microscope.

FIG. 32 is a scanning electron micrograph at 1000× magnification of afibrous material produced from shearing switchgrass on a rotary knifecutter, and then passing the sheared material through a 1/32 inchscreen.

FIGS. 33 and 34 are scanning electron micrographs of the fibrousmaterial of FIG. 32 after irradiation with 10 Mrad and 100 Mrad gammarays, respectively, at 1000× magnification.

FIG. 35 is a scanning electron micrographs of the fibrous material ofFIG. 32 after irradiation with 10 Mrad and sonication at 1000×magnification.

FIG. 36 is a scanning electron micrographs of the fibrous material ofFIG. 32 after irradiation with 100 Mrad and sonication at 1000×magnification.

Example 22 Fourier Transform Infrared (FT-IR) Spectrum of Irradiated andUnirradiated Kraft Paper

FT-IR analysis was performed on a Nicolet/Impact 400. The resultsindicate that samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e,P-70e, and P-100e are consistent with a cellulose-based material.

FIG. 37 is an infrared spectrum of Kraft board paper sheared accordingto Example 4, while FIG. 38 is an infrared spectrum of the Kraft paperof FIG. 37 after irradiation with 100 Mrad of gamma radiation. Theirradiated sample shows an additional peak in region A (centered about1730 cm-1 that is not found in the un-irradiated material. Of note, anincrease in the amount of a carbonyl absorption at 1650 cm-1 wasdetected when going from P132 to P132-10 to P132-100. Similar resultswere observed for the samples P-1e, P-5e, P-10e, P-30e, P-70e, andP-100e.

Example 23 Proton and Carbon-13 Nuclear Magnetic Resonance (¹H-NMR and¹³C-NMR) Spectra of Irradiated and Unirradiated Kraft Paper

Sample Preparation

The samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e, P-70e,and P-100e were prepared for analysis by dissolution with DMSO-d6 with2% tetrabutyl ammonium fluoride trihydrate. The samples that hadundergone lower levels of irradiation were significantly less solublethan the samples with higher irradiation. Unirradiated samples formed agel in this solvent mixture, but heating to 60° C. resolved the peaks inthe NMR spectra. The samples having undergone higher levels ofirradiation were soluble at a concentration of 10% wt/wt.

Analysis

¹H-NMR spectra of the samples at 15 mg/mL showed a distinct very broadresonance peak centered at I6 ppm (FIGS. 38A-38J). This peak ischaracteristic of an exchangeable —OH proton for an enol and wasconfirmed by a “d₂O shake.” Model compounds (acetylacetone, glucuronicacid, and keto-gulonic acid) were analyzed and made a convincing casethat this peak was indeed an exchangeable enol proton. This proposedenol peak was very sensitive to concentration effects, and we wereunable to conclude whether this resonance was due to an enol or possiblya carboxylic acid.

The carboxylic acid proton resonances of the model compounds weresimilar to what was observed for the treated cellulose samples. Thesemodel compounds were shifted up field to 5-6 ppm. Preparation of P-I00eat higher concentrations (I 0% wt/wt) led to the dramatic down fieldshifting to where the carboxylic acid resonances of the model compoundswere found (6 ppm) (FIG. 38N). These results lead to the conclusion thatthis resonance is unreliable for characterizing this functional group,however the data suggests that the number of exchangeable hydrogensincreases with increasing irradiation of the sample. Also, no vinylprotons were detected.

The 13C NMR spectra of the samples confirm the presence of a carbonyl ofa carboxylic acid or a carboxylic acid derivative. This new peak (at I68ppm) is not present in the untreated samples (FIG. 38K). A 13C NMRspectrum with a long delay allowed the quantitation of the signal forP-100e (FIGS. 38L-38M). Comparison of the integration of the carbonylresonance to the resonances at approximately 100 ppm (the C I signals)suggests that the ratio of the carbonyl carbon to C I is 1:13.8 orroughly 1 carbonyl for every 14 glucose units. The chemical shift at 100ppm correlates well with glucuronic acid.

Titration

Samples P-100e and P132-100 (1 g) were suspended in deionized water (25mL). The indicator alizarin yellow was added to each sample withstirring. P-100e was more difficult to wet. Both samples were titratedwith a solution of 0.2M NaOH. The end point was very subtle and wasconfirmed by using pH paper. The starting pH of the samples was 4 forboth samples. P132-100 required 0.4 milliequivalents of hydroxide, whichgives a molecular weight for the carboxylic acid of 2500 amu. If 180 amuis used for a monomer, this suggests there is one carboxylic acid groupfor 13.9 monomer units. Likewise, P-100e required 3.2 milliequivalentsof hydroxide, which calculates to be one carboxylic acid group for every17.4 monomer units.

Conclusions

The C-6 carbon of cellulose appears to be oxidized to the carboxylicacid (a glucuronic acid derivative) in this oxidation is surprisinglyspecific. This oxidation is in agreement with their band that grows withirradiation at 1740 cm-¹ which corresponds to an aliphatic carboxylicacid. The titration results are in agreement with the quantitative ¹³CNMR. The increased solubility of the sample with the higher levels ofirradiation correlates well with the increasing number of carboxylicacid protons. A proposed mechanism for the degradation of “C-6 oxidizedcellulose” is provided below in Scheme 1.

Example 24 Combination of Electron Beam and Sonication Pretreatment

Switchgrass is used as the feedstock and is sheared with a Munson rotaryknife cutter into a fibrous material. The fibrous material is thenevenly distributed onto an open tray composed of tin with an area ofgreater than about 500 in2. The fibrous material is distributed so thatit has a depth of about 1-2 inches in the open tray. The fibrousmaterial may be distributed in plastic bags at lower doses ofirradiation (under 10 MRad), and left uncovered on the metal tray athigher doses of radiation.

Separate samples of the fibrous material are then exposed to successivedoses of electron beam radiation to achieve a total dose of 1 Mrad, 2Mrad, 3, Mrad, 5 Mrad, 10 Mrad, 50 Mrad, and 100 Mrad. Some samples aremaintained under the same conditions as the remaining samples, but arenot irradiated, to serve as controls. After cooling, the irradiatedfibrous material is sent on for further processing through a sonicationdevice.

The sonication device includes a converter connected to boostercommunicating with a horn fabricated from titanium or an alloy oftitanium. The horn, which has a seal made from VITON® about itsperimeter on its processing side, forms a liquid tight seal with aprocessing cell. The processing side of the horn is immersed in aliquid, such as water, into which the irradiated fibrous material to besonicated is immersed. Pressure in the cell is monitored with a pressuregauge. In operation, each sample is moved by pump through the processingcell and is sonicated.

To prepare the irradiated fibrous material for sonication, theirradiated fibrous material is removed from any container (e.g., plasticbags) and is dispersed in water at a concentration of about 0.10 g/mL.Sonication is performed on each sample for 30 minutes using 20 kHzultrasound from a 1000 W horn under re-circulating conditions. Aftersonication, the irradiated fibrous material is captured in a tank. Thisprocess can be repeated a number of times until a desired level ofprocessing is achieved based on monitoring the structural changes in theswitchgrass. Again, some irradiated samples are kept under the sameconditions as the remaining samples, but are not sonicated, to serve ascontrols. In addition, some samples that were not irradiated aresonicated, again to serve as controls. Thus, some controls are notprocessed, some are only irradiated, and some are only sonicated.

Example 25 Microbial Testing of Pretreated Biomass

Specific lignocellulosic materials pretreated as described herein areanalyzed for toxicity to common strains of yeast and bacteria used inthe biofuels industry for the fermentation step in ethanol production.Additionally, sugar content and compatibility with cellulase enzymes areexamined to determine the viability of the treatment process. Testing ofthe pretreated materials is carried out in two phases as follows.

Phase 1: Toxicity and Sugar Content

Toxicity of the pretreated grasses and paper feedstocks is measured inyeast Saccharomyces cerevisiae (wine yeast) and Pichia stipitis (ATCC66278) as well as the bacteria Zymomonas mobilis (ATCC 31821) andClostridium thermocellum (ATCC 31924). A growth study is performed witheach of the organisms to determine the optimal time of incubation andsampling.

Each of the feedstocks is then incubated, in duplicate, with S.cerevisiae, P. stipitis, Z. mobilis, and C. thermocellum in a standardmicrobiological medium for each organism. YM broth is used for the twoyeast strains, S. cerevisiae and P. stipitis. RM medium is used for Z.mobilis and CM4 medium for C. thermocellum. A positive control, withpure sugar added, but no feedstock, is used for comparison. During theincubation, a total of five samples is taken over a 12 hour period attime 0, 3, 6, 9, and 12 hours and analyzed for viability (plate countsfor Z. mobilis and direct counts for S. cerevisiae) and ethanolconcentration.

Sugar content of the feedstocks is measured using High PerformanceLiquid Chromatography (HPLC) equipped with either a Shodex™ sugar SP0810or Biorad Aminex® HPX-87P column. Each of the feedstocks (approx. 5 g)is mixed with reverse osmosis (RO) water for 1 hour. The liquid portionof the mixture is removed and analyzed for glucose, galactose, xylose,mannose, arabinose, and cellobiose content. The analysis is performedaccording to National Bioenergy Center protocol Determination ofStructural Carbohydrates and Lignin in Biomass.

Phase 2: Cellulase Compatibility

Feedstocks are tested, in duplicate, with commercially availableAccellerase™ 1000, which contains a complex of enzymes that reduceslignocellulosic biomass into fermentable sugars, at the recommendedtemperature and concentration in an Erlenmeyer flask. The flasks areincubated with moderate shaking at around 200 rpm for 12 hours. Duringthat time, samples are taken every three hours at time 0, 3, 6, 9, and12 hours to determine the concentration of reducing sugars (Hope andDean, Biotech J., 1974, 144:403) in the liquid portion of the flasks.

Example 26 Sugar Concentration Analysis Using HPLC

13 samples were analyzed for sugar concentration (HPLC) and toxicityagainst 3 microorganisms (Pichia stipitis, Saccharomyces cerevisiae, andZymomonas mobilis. Table 26 lists the equipment used for theseexperiments. Table 27 and 28 provide a list of the sugars (includingvendor and lot numbers) used to prepare the HPLC standard and theprotocol used to prepare the HPLC standard, respectively.

TABLE 26 Equipment Utilized in Experiments Equipment Manufacturer, NamepH meter Orion Shakers (2) B. Braun Biotech, Certomat BS-1 HPLC Waters,2690 HPLC Module Spectrophotometer Unicam, UV300 YSI Biochem AnalyzerInterscience YSI

TABLE 27 Sugars used in HPLC analysis Sugar Manufacturer Ref# Lot#glucose BioChemika 49140 1284892 xylose 95731 1304473 51707231cellobiose 22150 1303157 14806191 arabinose 10840 1188979 24105272mannose 63582 363063/1 22097 galactose 48259 46032/1 33197

TABLE 28 Preparation of HPLC standards Volume of Total DesiredConcentration Volume of sugar Nanopure Water Volume (mg/ml) solution(ml) (ml) 4 50 ml of 4 mg/ml 0 50 2 25 ml of 4 mg/ml 25 50 1 25 ml of 2mg/ml 25 50 0.5 25 ml of 1 mg/ml 25 50 0.1 5 ml of 1 mg/ml 20 25Verification 18.75 ml of 4 31.25 50 Standard 1.5 mg/mL

Analysis

Each sample (1 gram) was mixed with reverse osmosis water at 200 rpm and50° C. overnight. The pH of the sample was adjusted to between 5 and 6and filtered through a 0.2) lm syringe filter. Samples were stored at−20° C. prior to analysis to maintain integrity of the samples. Theobservations made during the preparation of the samples are presented inTable 29.

TABLE 29 Observations During HPLC Sample Preparation Amount Used Wateradded Sample (g) (mL) pH Observations P132 30 5.38 Fluffy, difficult tomix P132-10 1 25 6.77 Fluffy, difficult to mix P132-100 1 20 3.19 pH islow, difficult to bring P132-US 0.3 5 6.14 to pH 5.0, used 10N NaOH A1321 15 5.52 A132-10 1 15 4.9 A132-100 1 15 5.39 SG132 1 15 5.59 SG132-10 115 5.16 SG132-100 1 15 4.7 SG132-10- 0.3 5 5.12 US S132-100- 0.3 5 4.97US WS132 1 15 5.63 WS132-10 1 15 5.43 WS132-100 1 15 5.02

Standards were prepared fresh from a 4 mg/mL stock solution of the 6combined sugars, glucose, xylose, cellobiose, arabinose, mannose, andgalactose. The stock solution was prepared by dissolving 0.400 grams ofeach sugar into 75 mL of nanopure water (0.3 micron filtered). Oncedissolved, the stock solution was diluted to 100 mL using a volumetricflask and stored at −20° C. Working standard solutions of 0.1, 0.5, 1,2, and 4 mg/mL were prepared by serial dilution of the stock solutionwith nanopure water. In addition, a verification standard of 1.5 mg/mLwas also prepared from the stock solution.

Sugar concentrations were analyzed according to the protocolDetermination of Structural Carbohydrates in Biomass (NREL BiomassProgram, 2006) and this protocol is incorporated herein by reference inits entirety. A SHODEX SUGAR SP0810 COLUMN with an Evaporative LightScattering Detector was used. A verification standard (1.5 mg/mL ofstandard) was analyzed every 8 injections to ensure that the integrityof the column and detector were maintained during the experiment. Thestandard curve coefficient of variation (R2 value) was at least 0.989and the concentration of the verification standards were within 10% ofthe actual concentration. The HPLC conditions were as follows:

TABLE 30 HPLC Parameters Injection volume 20 μL Mobile phase: NanopureWater*, 0.45 μm Filtered and degassed Flow rate: 0.5 mL/min Column 85°C. Temperature: Detector Evaporator temperature Temperature: 110° C.,nebulizer Temperature 90° C. *Initial tests noted that better separationwas observed when using nanopure water than 15/85 acetonitrile:water inthe mobile phase (manufacturer does not recommend using greater than 20%acetonitrile with this column).

Results

The results of the HPLC analysis are presented in Tables 31, 32, and 33.

TABLE 31 Sugar Concentration Expressed as mg/mL and mg/g of ExtractXylose Arabinose Glucose Cellobiose mW-150 mW-150 mW-180 GalactoseMannose mW-342 C₅H₁₀O₅ C₅H₁₀O₅ C₆H₁₂O₆ (see glue) (see glue) C₁₂H₂₂O₁₁Sample Mono Mono Mono mg/mL: mg/g mg/mL: mg/g Disacc ID mg/mL mg/g mg/mLmg/g mg/mL mg/g mg/mL mg/g mg/mL mg/g mg/mL mg/g P P-132 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 P-132-10 0.00 0.00 0.000.00 0.34 8.60 0.00 0.00 0.00 0.00 00.33 8.13 P-132-100 0.35 7.04 0.000.00 0.34 6.14 0.00 0.00 0.00 0.00 0.36 7.20 P-132-BR 0.35 5.80 0.437.17 0.34 5.62 0.00 0.00 0.00 0.00 0.00 0.00 G G-132 0.39 5.88 0.38 5.730.84 12.66 0.34 5.04 0.92 13.76 0.00 0.00 G-132-10 0.50 7.50 0.41 6.181.07 16.04 0.35 5.19 0.98 14.66 0.00 0.00 G-132-100 0.00 0.00 0.37 5.540.41 6.14 0.00 0.00 0.55 8.28 0.45 6.71 G-132-10-US 0.34 5.73 0.39 6.450.33 5.43 0.00 0.00 0.00 0.00 0.00 0.00 G-132-100-US 0.00 0.00 0.37 6.220.35 5.90 0.33 5.43 0.40 6.70 0.39 6.45 A A-132 1.36 20.39 0.00 0.001.08 16.22 0.39 5.84 1.07 16.02 0.00 0.00 A-132-10 1.19 17.87 0.00 0.000.00 0.00 0.00 0.00 0.37 5.52 0.00 0.00 A132-100 1.07 16.11 0.00 0.000.35 5.18 0.00 0.00 0.00 0.00 0.81 12.2 WS WS-132 0.49 7.41 0.41 6.150.39 5.90 0.00 0.00 0.00 0.00 0.00 0.00 WS-132-10 0.57 8.49 0.40 5.990.73 10.95 0.34 5.07 0.50 7.55 0.00 0.00 WS-132-100 0.43 6.39 0.37 5.510.36 5.36 0.00 0.00 0.36 5.33 0.35 5.25

TABLE 32 Sugar Concentration Expressed at % of Paper Sugar concentration(% of dry sample) P132 P132-10 P132-100 P132-US cellobiose 0.00 0.810.72 0.00 glucose 0.00 0.86 0.67 0.56 xylose 0.00 0.00 0.70 0.58galactose 0.00 0.00 0.00 0.00 arabinose 0.00 0.00 0.00 0.72 mannose 0.000.00 0.00 0.00

TABLE 33 Sugar Concentration Expressed at % of Total Sample A132- A132-SG132- SG132- SG132- SG132- WS132- WS132- Sugar A132 10 100 SG132 10 1010-US 100-US WS132 10 100 cellobiose 0.00 0.00 1.22 0.00 0.00 0.67 0.000.65 0.00 0.00 0.53 glucose 1.62 0.00 0.52 1.27 1.60 0.61 0.54 0.59 0.591.10 0.54 xylose 2.04 1.79 1.61 0.59 0.75 0.00 0.57 0.00 0.74 0.85 0.64galactose 0.58 0.00 0.00 0.50 0.52 0.00 0.00 0.54 0.00 0.51 0.00arabinose 0.00 0.00 0.00 0.57 0.62 0.55 0.65 0.62 0.62 0.60 0.55 mannose1.60 0.55 0.00 1.38 1.47 0.83 0.00 0.67 0.00 0.76 0.53

Example 27 Toxicity Study

Twelve samples were analyzed for toxicity against a panel of threeethanol-producing cultures. In this study, glucose was added to thesamples in order to distinguish between starvation of the cultures andtoxicity of the samples. A thirteenth sample was tested for toxicityagainst Pichia stipitis. A summary of the protocol used is listed inTable 32. A description of the chemicals and equipment used in thetoxicity testing is reported in Tables 34-36.

TABLE 34 Conditions for Toxicity Testing Organism Pichia ZymomonasSaccharomyces stipites mobilis cerevesiae NRRL Variable ATCC 31821 ATCC24858 Y-7124 Test Repetition Duplicate Inoculation Volume (mL)  1   0.1 1 Incubation Temperature 30° C. 25° C. 25° C. Shaker Speed (rpm) 125 200  125  Erlenmeyer Flask Volume 250 mL 500 mL 250 mL Media volume 100mL 100 mL 100 mL Total Incubation time 36 36 48 (hours) Ethanol Analysis(hours) 24, 30, 36 24, 30, 36 24, 36, 48 Cell Counts (hours) 24, 36 24,36 24, 36 pH 0 hours 0 hours 0 hours

TABLE 35 Reagents Used for Toxicity Testing Media Component ManufacturerReference # Lot # Urea Yeast ScholAR chemistry 9472706 AD-7284-43Nitrogen Base Becton Dickinson 291940 7128171 Peptone Becton Dickinson211677 4303198 Xylose Fluka 95731 1304473 51707231 Glucose Yeast ExtractSigma G-5400 107H0245 (used for S. cerevisiae) Becton Dickinson 2886204026828 Yeast Extract (used for Becton Dickinson 212750 7165593 Pstipites and Z. mobilis) MgS04 Sigma M5921 034K0066 7H₂O Sigma A4418117K5421 (NH₄)₂S0₄ P5379 074K0160 KH₂P0₄ 271120 6278265

TABLE 36 YSI Components Used in Shake Flask Study Component Catalog #Lot # YSI Ethanol Membrane 2786 07L100153 YSI Ethanol Standard (3.2 g/L)2790 012711040 YSI Ethanol Buffer 2787 07M1000053, 07100215

Testing was performed using the three microorganisms as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

A slant of S. cerevisiae was prepared from a rehydrated lyophilizedculture obtained from ATCC. A portion of the slant material was streakedonto an YM Broth+20 g/L agar (pH 5.0) and incubated at 30° C. for 2days. A 250 mL Erlenmeyer flask containing 50 mL of medium (20 g/Lglucose, 3 g/L yeast extract, and 5.0 giL peptone, pH 5.0) wasinoculated with one colony from the YM plate and incubated for 24 hoursat 25° C. and 200 rpm. After 23 hours of growth, a sample was taken andanalyzed for optical density (600 nm in a UV spectrophotometer) andpurity (Gram stain). Based on these results, one flask (called the SeedFlask) with an OD of 14.8 and clean Gram Stain was chosen to inoculateall of the test flasks.

The test vessels were 500 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved at 121° C.and 15 psi prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving will change the content ofthe samples. The test samples were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 1 mL (1% v/v) of seed flask material wasadded to each flask. The flasks were incubated as described above for 36hours.

Pichia stipitis NRRL Y-7124 (ARS Culture Collection)

A slant of P. stipitis was prepared from a rehydrated lyophilizedculture obtained from ARS Culture Collection. A portion of the slantmaterial was streaked onto an YM Broth+20 g/L agar (pH 5.0) andincubated at 30° C. for 2 days. A 250 mL Erlenmeyer flask containing 100mL of medium (40 g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/Lurea, 6.56 g/L peptone, 40 g/L xylose, pH 5.0) was inoculated with asmall amount of plate material and incubated for 24 hours at 25° C. and125 rpm. After 23 hours of growth, a sample was taken and analyzed foroptical density (600 nm in a UV spectrophotometer) and purity (Gramstain). Based on these results, one flask (called the Seed Flask) at anoptical density of 5.23 and with a clean Gram Stain was chosen toinoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved empty at 121°C. and 15 psi and filter sterilized (0.22) 1 m filter) media added tothe flasks prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving will change the content ofthe samples and filter sterilization not appropriate for sterilizationof solids. The test samples were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 1 mL (1% v/v) of seed flask material wasadded to each flask. The flasks were incubated as described above for 48hours.

Zymomonas mobilis ATCC 31821 (American Type Culture)

A slant of Z. mobilis was prepared from a rehydrated lyophilized cultureobtained from ATTC. A portion of the slant material was streaked ontoDYE plates (glucose 20 g/L, Yeast Extract 10 g/L, Agar 20 g/L, pH 5.4)and incubated at 30° C. and 5% CO₂ for 2 days. A 20 mL screw-cap testtube containing 15 mL of medium (25 g/L glucose, 10 g/L yeast extract, 1g/L MgSO₄, 7H₂O, 1 g/L (NH₄)2SO₄, 2 g/L KH₂PO₄, pH 5.4) was inoculatedwith one colony and incubated for 24 hours at 30° C. with no shakingAfter 23 hours of growth, a sample was taken and analyzed for opticaldensity (600 nm in a UV spectrophotometer) and purity (gram stain).Based on these results, one tube (OD 1.96) was chosen to inoculate thesecond seed flask. The second seed flask was a 125 ml flask containing70 mL of the media described above and was inoculated with 700 mL (1%v/v) and incubated for 24 hours at 30° C. with no shaking After 23 hoursof growth, a sample was taken and analyzed for optical density (600 nmin a UV spectrophotometer) and purity (gram stain). Based on theseresults, one flask (called the Seed Flask) with an OD of 3.72 was chosento inoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above with the exception of yeast extract at 5g/L. All flasks were autoclaved empty at 121° C. and 15 psi and filtersterilized (0.22) μm filter) media added to the flasks prior to theaddition of the test materials. The test materials were not sterilized,as autoclaving will change the content of the samples and filtersterilization not appropriate for sterilization of solids. The testsamples were added at the time of inoculation to reduce the possibilityof contamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated asdescribed above for 36 hours

Analysis

Two samples were analyzed for cell concentration (using spread platingfor Z. mobilis and direct counts (haemocytometer and microscope for S.cerevisiae and P. stipitis). Appropriately diluted samples of Z. mobiliswere spread on Dextrose Yeast Extract (glucose 20 g/L, Yeast Extract 10g/L, Agar 20 g/L, pH 5.4) plates, incubated at 30° C. and 5% CO2 for 2days, and the number of colonies counted. Appropriately diluted samplesof S. cerevisiae and P. stipitis were mixed with 0.05% Trypan blue,loaded into a Neubauer haemocytometer. The cells were counted under 40×magnification.

Three samples were analyzed for ethanol concentration using the YSIBiochem Analyzer based on the alcohol dehydrogenase assay (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. to preserve integrity. The sampleswere diluted to between 0-3.2 g/L ethanol prior to analysis. A standardof 3.2 g/L ethanol was analyzed approximately every 30 samples to ensurethe integrity of the membrane was maintained during analysis. Theoptical density (600 nm) of the samples is not reported because thesolid test samples interfered with absorbance measurement by increasingthe turbidity of the samples and are inaccurate.

Results of Ethanol Analysis

Performance was used to compare each sample to the control for eachmicroorganism (Tables 37-39). However, the % performance cannot be usedto compare between strains. When comparing strains, the totalconcentration of ethanol should be used. When analyzing the data, a %performance of less than 80% may indicate toxicity when accompanied bylow cell number. The equation used to determine % performance is:% Performance=(ethanol in the sample/ethanol in control)×100

TABLE 37 Ethanol Concentration and % Performance Using Saccharomycescerevisiae 24 hours 30 hours 36 hours Ethanol Ethanol EthanolConcentration % Concentration % Concentration % Sample# (g/L)Performance (g/L) Performance (g/L) Performance P132 4.0 140 5.2 1273.26 176 P132-10 4.2 147 5.1 125 3.86 209 P132-100 4.3 149 5.6 136 3.47187 A132 5.5 191 6.5 160 5.24 283 A132-10 1.9 67 6.3 153 5.54 299A132-100 4.4 154 5.6 137 4.04 218 G132 5.3 186 6.0 146 3.99 215 G132-105.2 180 6.4 156 4.63 250 G132-100 5.5 191 6.3 155 4.60 248 WS132 4.8 1686.3 155 4.51 244 WS132-10 4.9 172 6.0 146 4.55 246 WS132-100 4.9 170 5.7140 4.71 254 Control 2.9 100 4.1 100 1.85 100

TABLE 38 Ethanol Concentration and % Performance Using Pichia stipitis24 hours 36 hours 48 hours Ethanol Ethanol Ethanol Concentration %Concentration % Concentration % Sample# (g/L) Performance (g/L)Performance (g/L) Performance P132 2.8 130 3.4 188 8.1 176 P132-10 7.3344 11.9 655 15.8 342 P132-100 5.2 247 8.6 472 13.3 288 A132 12.2 57514.7 812 14.9 324 A132-10 15.1 710 18.7 1033 26.0 565 A132-100 10.9 51416.7 923 22.2 483 G132 8.0 375 12.9 713 13.3 288 G132-10 10.1 476 16.0884 22.3 485 G132-100 8.6 406 15.2 837 21.6 470 WS132 9.8 460 14.9 82017.9 389 WS132-10 7.8 370 16.1 890 19.3 418 WS132-100 9.1 429 15.0 82915.1 328 Sample A* 13.2 156 19.0 166 20.6 160 Control 2.1 100 1.8 1004.6 100 Samples m BOLD were the highest ethanol producers, over 20 g/Land similar to the concentrations in wood hydrolyzates m (H. K. Sreenathand T. W. Jeffries Bioresource Technology 72 (2000) 253-260). *Analyzedin later shake flask experiment.

TABLE 39 Ethanol Concentration and % Performance Using Zymomonas mobilis24 hours 30 hours 36 hours Ethanol Ethanol Ethanol Concentration %Concentration % Concentration % Sample# (g/L) Performance (g/L)Performance (g/L) Performance P132 7.5 85 6.8 84 7.5 93 P132-10 7.5 854.8 59 6.8 84 P132-100 7.3 83 6.2 77 7.1 88 A132 9.6 109 8.3 103 9.1 112A132-10 9.2 105 8.4 105 8.8 109 A132-100 8.2 93 7.6 94 7.6 93 WS132 7.989 7.1 88 7.7 94 WS132-10 8.2 93 6.8 85 7.3 90 WS132-100 8.7 98 6.9 868.3 102 G132 8.7 99 7.1 88 8.1 99 G132-10 7.8 88 7.0 88 7.3 90 G132-1008.6 98 7.8 98 8.3 102 Control 8.8 100 8.0 100 8.1 100

Results from Cell Concentration Analysis

-   -   % Cells is used to compare each sample to the control for each        organism (Tables 40-42). However, the % cells cannot be used to        compare between strains. When comparing strains, the total        concentration of cells should be used. When analyzing the data,        a % performance of less than 70% may indicate toxicity when        accompanied by low ethanol concentration. The equation used to        determine % performance is:        % cells=(number of cell in the sample/number of cells in        control)×100

TABLE 40 Results from Cell Concentration Analysis for Saccharomycescerevisiae 24 hours 36 hours Cell Cell Sample# Concentration % CellsConcentration % Cells P132 1.99 166 2.51 83 P132-10 2.51 209 1.91 63P132-100 1.35 113 1.99 66 A132 3.80 316 2.59 85 A132-10 1.73 144 3.90129 A132-100 3.98 331 2.51 83 G132 2.14 178 3.12 103 G132-10 2.33 1942.59 85 G132-100 3.57 298 2.66 88 WS132 4.10 341 2.66 88 WS132-10 2.63219 2.81 93 WS132-100 2.29 191 2.40 79 Control 1.20 100 3.03 100

TABLE 41 Results from Cell Concentration Analysis for Pichia stipitis 24hours 48 hours Cell Cell Sample# Concentration ( % Cells Concentration (% Cells P132 16.4 108 20.3 87 P132-10 11.5 76 9.5 41 P132-100 6.5 4317.8 76 A132 7.1 47 10.2 44 A132-10 12.7 84 9.3 40 A132-100 11.8 78 18.378 G132 4.5 30 4.8 21 G132-10 22.8 151 9.8 42 G132-100 10.1 67 21.7 93WS132 17.6 117 8.2 35 WS132-10 5.3 35 10.8 46 WS132-100 9.3 62 10.7 46Control 15.1 100 23.4 100

TABLE 42 Results from Cell Concentration Analysis for Zymomonas mobilis24 hours 36 hours Cell Cell Sample# Concentration ( % CellsConcentration ( % Cells P132 7.08 86 2.97 66 P132-10 21.80 264 4.37 98P132-100 4.50 54 3.35 75 A132 6.95 84 1.99 44 A132-10 6.13 74 4.05 91A132-100 9.60 116 4.20 94 G132 7.48 90 3.84 86 G132-10 14.75 178 2.89 65G132-100 6.00 72 2.55 57 WS132 9.70 117 4.55 102 WS132-10 13.20 160 4.3297 WS132-100 5.15 62 2.89 65 Control 8.27 100 4.47 100

Example 28 Shake Flask Fermentation of Cellulose Samples Using P.stipitis

Summary

Thirteen samples were tested for ethanol production in P. stipitisculture without sugar added. They were tested in the presence andabsence of cellulase (Accellerase 1000® enzyme complex, Genencor).Equipment and reagents used for the experiment are listed below inTables 43-45.

TABLE 43 Equipment and frequency of maintenance Equipment ManufacturerB. Frequency of Maintenance Braun Biotech, Shakers (2) Certomat BS-1Quartlerly Unicam, UV300 Spectrophotometer Interscience, YSI BiannualYSI Biochem Analyzer Monthly

TABLE 44 YSI Components used in shake flask study Component Catalog# Lot# YSI Ethanol Membrane 2786 07L100153 YSI Ethanol Standard (3.2 279002711040 g/L) YSI Ethanol Buffer 2787 07M1000053, 07100215

TABLE 45 Chemicals used for shake flask fermentation Media ComponentManufacturer Reference # Lot # ScholAR 9472706 AD-7284-43 Urea Chemistry7128171 Yeast Nitrogen Becton Dickinson 291940 Base Peptone BectonDickinson 211677 4303198 YM Broth Becton Dickinson 271120 6278265Accellerase ® Genoncor Accellerase ® 1600794133 Enzyme Complex 1000Xylose BioChemika 95731 1304473 51707231 Glucose Sigma G-5400 107H0245

A slant of P. stipitis NRRL Y-7124 was prepared from a rehydratedlyophilized culture obtained from ARS Culture Collection. A portion ofthe slant material was streaked onto a Yeast Mold (YM) Broth+20 g/L agar(pH 5.0) and incubated at 30° C. for 2 days. A 250 mL Erlenmeyer flaskcontaining 100 mL of medium (40 g/L glucose, 1.7 g/L yeast nitrogenbase, 2.27 g/L urea, 6.56 g/L peptone, 40 g/L xylose, pH 5.0) wasinoculated with one colony and incubated for 24 hours at 25° C. and 100rpm. After 23 hours of growth, a sample was taken and analyzed foroptical density (600 nm in a UV spectrophotometer) and purity (Gramstain). Based on these results, one flask (called the Seed Flask) at anoptical density of 6.79 and with a clean Gram stain was chosen toinoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL ofmedium (1.7 g/L yeast nitrogen base, 2.27 g/L urea, and 6.56 giLpeptone). No sugar (glucose or xylose) was added to the growth flaskmedium. All flasks were autoclaved empty at 121° C. and 15 psi andfilter sterilized (0.22) lm filter) media added to the flasks prior tothe addition of the test materials. The test materials were notsterilized, as autoclaving will change the content of the samples andfilter sterilization is not appropriate for sterilization of solids. Thetest samples (listed in Table 46) were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 1 mL (1% v/v) of seed flask material wasadded to each flask. Flasks containing sample P132-100 required theaddition of 0.4 mL 1 M NaOH to bring the pH to 5.0. The flasks wereincubated at 30° C. and 150 rpm above for 96 hours.

One set of duplicate flasks per feedstock contained Accellerase® enzymecomplex (1.25 mL per flask, highest recommended dosage is 0.25 mL pergram of biomass, Genencor) to attempt simultaneous saccharification andfermentation (SSF). The other set of duplicate flasks did not containAccellerase® enzyme complex. A total of 52 flasks were analyzed.

Six control flasks were also analyzed. Positive control flasks containedSolkaFloc 200 NF Powdered Cellulose (lot #UA158072, International FiberCorporation) at a concentration of 2.5 grams per 100 mL flask (25 gramsper L) with and without addition of Accellerase® enzyme complex. Inaddition, a control containing sugars (glucose and xylose) only wasused.

TABLE 46 The amount of each feedstock added to each flask Xyleco AmountAdded to Flask Number (g/100 ml) P132 2.5 P132-10 2.5 P132-100 2.5 A1325 A132-10 5 A132-100 5 G132 5 G132-10 5 G132-100 5 WS132 5 WS132-10 5WS132-100 5 Sample A 5

Analysis

Samples were analyzed for ethanol concentration (Tables 47, 48, and 49)using the YSI Biochem Analyzer based on the alcohol dehydrogenase assay(YSI, Interscience). Samples were centrifuged at 14,000 rpm for 20minutes and the supernatant stored at −20° C. The samples were dilutedto between 0-3.2 g/L ethanol prior to analysis. A standard of 2.0 g/Lethanol was analyzed approximately every 30 samples to ensure theintegrity of the membrane was maintained during analysis.

Results

TABLE 47 Results of Control Flasks Ethanol Concentration (g/L) Control24 hours 36 hours 48 hours 96 hours Containing Glucose, no 13.20 19.0020.60 21.60 cellulose, no enzyme Containing Crystalline 0.00 0.00 0.000.00 Cellulose (Solka Floc), Containing Crystalline 6.56 7.88 9.80 8.65Cellulose (Solka Floc) at 25 g/L, no sugar, Accellerase ® added

TABLE 48 Results of Shake Flasks without Accellerase ® 1000 EnzymeComplex Sample Ethanol Concentration (g/L) Number 24 hours 36 hours 48hours 96 hours P132 0.09 0.00 0.00 0.12 P132-10 0.02 0.01 0.02 0.17P132-100 0.09 0.01 0.00 0.02 A132 1.74 1.94 2.59 3.70 A132-10 1.82 2.362.30 2.96 A132-100 0.30 0.73 1.31 2.38 G132 0.40 0.09 0.24 0.42 G132-100.69 0.42 0.22 0.24 G132-100 0.19 0.05 0.05 0.21 WS132 0.47 0.50 0.680.65 WS132-10 0.47 0.49 0.34 0.92 WS132-100 0.14 0.07 0.08 0.22 Sample A1.88 1.89 2.30 3.28

TABLE 49 Results of Shake Flasks with Accellerase ® 1000 Enzyme ComplexSample Ethanol Concentration (g/L) Number 24 hours 36 hours 48 hours 96hours P132 7.04 8.72 9.30 5.80 P132-10 4.22 4.48 4.49 1.24 P132-100 3.184.28 4.70 3.35 A132 2.79 2.91 2.03 4.30 A132-10 3.31 1.62 2.11 2.71A132-100 2.06 1.92 1.02 1.47 G132 0.87 0.40 0.32 0.44 G132-10 1.38 1.040.63 0.07 G132-100 2.21 2.56 2.34 0.12 WS132 1.59 1.47 1.07 0.99WS132-10 1.92 1.18 0.73 0.23 WS132-100 2.90 3.69 3.39 0.27 Sample A 2.212.35 3.39 2.98

Example 29 Cellulase Assay

Summary

Thirteen samples were tested for cellulase susceptibility using anindustry cellulase (Accellerase® 1000, Genencor) under optimumconditions of temperature and pH.

Protocol

The protocol is a modification of the NREL “Laboratory AnalyticalProcedure LAP-009 Enzymatic Saccharification of LignocellulosicBiomass”. A sample of material was added to 10 mL 0.1 M sodium citratebuffer (pH 4.8) and 40 mg/mL tetracycline (to prevent growth ofbacteria) in a 50 mL tube in duplicate. The amount of sample added toeach tube is listed in Table 50. Some samples were difficult to mix(P132, P132-10, P132-100), so were added at a lower concentration. Apositive control of 0.2 grams SolkaFloc 200 NF Powdered Cellulose (lot#UA158072, International Fiber Corporation) and a negative control (nosample) were also included. Enough reverse osmosis (RO) water to bringthe volume to a total of 20 mL was added to the tubes. Both the sodiumcitrate buffer and water were heated to 50° C. prior to use.

Accellerase® 1000 enzyme was added to each tube at a dosage of 0.25 mLper gram of biomass (highest dosage recommended by Genencor). The tubeswere incubated at 45° angle at 150 rpm and 50° C. (recommended byGenencor) for 72 hours. Samples were taken at 0, 3, 6, 9, 12, 18, 24,48, and 72 hours (Table 52 and 53), centrifuged at 14,000 rpm for 20minutes and the supernatant frozen at −20° C. The glucose concentrationin the samples was analyzed using the YSI Biochem Analyzer(Interscience) using the conditions described in Table 51. A glucosestandard solution of 2.5 g/L was prepared by dissolving 2.500 gramsglucose (Sigma Cat #G7528-5KG, Lot #:107H0245) in distilled water. Oncedissolved, the total volume was brought to 1 L with distilled water in avolumetric flask. The standard was prepared fresh weekly and stored at4° C.

TABLE 50 Amount of Each Sample Added Xyleco Amount added to Tube Number(g/20 ml) P132 0.5 P132-10 0.5 P132-100 0.5 A132 0.75 A132-10 0.75A132-100 0.75 G132 0.75 G132-10 0.75 G132-100 0.75 WS132 0.75 WS132-100.75 WS132-100 0.75 Sample A 0.75 SolkaFloc 200NF 0.2 (Control) NegativeControl 0

TABLE 51 YSI Components Used in Shake Flask Study Component Catalog #Lot # YSI Gluclose Membrane 2365 070100124 YSI Glucose Buffer 2357014614A

Results

TABLE 52 Cellulase Assay Results Sample Glucose Concentration (mg/ml) atIncubation Time (hours) Number 0 3 6 9 12 18 24 48 72 P132 0.59 4.197.00 8.72 9.70 10.95 12.19 15.10 15.65 P132-10 0.36 3.37 5.08 6.39 6.987.51 8.99 11.25 11.65 P132-100 0.91 3.86 5.67 7.31 8.08 9.47 10.70 12.7013.80 A132 0.39 1.51 1.92 2.40 2.64 3.04 3.30 3.90 4.06 A132-10 0.421.80 2.27 2.63 2.86 3.16 3.43 4.02 4.14 A132-100 0.46 2.09 2.72 3.163.43 3.78 4.09 4.84 5.26 G132 0.40 1.16 1.35 1.52 1.60 1.67 1.85 2.102.21 G132-10 0.34 1.34 1.64 1.95 2.03 2.09 2.36 2.77 3.02 G132-100 0.611.84 2.32 2.89 3.14 3.52 3.97 4.81 5.44 WS132 0.35 1.48 1.81 2.14 2.262.50 2.70 3.18 3.26 WS132-10 0.44 1.77 2.22 2.60 2.76 2.61 3.15 3.623.82 WS132-100 0.70 2.76 3.63 4.59 4.78 5.29 5.96 6.99 7.43 Sample A0.42 1.09 1.34 1.55 1.69 1.66 2.17 2.96 3.71 Negative Control 0.03 0.030.01 0.01 0.02 0.01 0.02 0.02 0.02 (no sample) Positive Control 0.172.38 3.65 4.71 5.25 5.98 7.19 9.26 9.86 (SolkaFloc)

Plots of glucose concentration (g/L) vs. time (hours) for the top fourproducers in Table 52 are shown in FIG. 39.

The amount of cellulose digested in the tube was calculated as follows:g/mL glucose×20mL(volume of sample)×0.9(to correct for the watermolecule added upon hydrolysis of cellulose)

The percent of the total sample released as glucose (in Table 53 below)was calculated as follows:g of cellulose digested/g of sample added(see Table 5 for details)*100

TABLE 53 Cellulase Assay Results Sample Percent of the Total SampleReleased as Glucose(%) at Incubation Time (h) Number 0 3 6 9 12 18 24 4872 P132 2.02 14.98 25.16 31.36 34.85 39.38 43.81 54.29 56.27 P132-101.19 12.02 18.25 22.97 25.06 27.00 32.29 40.43 41.87 P132-100 3.17 13.7920.38 26.28 29.02 34.06 38.45 45.65 49.61 A132 0.86 3.55 4.58 5.74 6.297.27 7.87 9.31 9.70 A132-10 0.94 4.25 5.42 6.29 6.82 7.56 8.18 9.60 9.89A132-100 1.03 4.94 6.50 7.56 8.18 9.05 9.77 11.57 12.58 G132 0.89 2.713.22 3.62 3.79 3.98 4.39 4.99 5.26 G132-10 0.74 3.14 3.91 4.66 4.82 4.995.62 6.60 7.20 G132-100 1.39 4.34 5.54 6.91 7.49 8.42 9.48 11.50 13.01WS132 0.77 3.48 4.32 5.11 5.38 5.98 6.43 7.58 7.78 WS132-10 0.98 4.185.30 6.22 6.58 6.24 7.51 8.64 9.12 WS132-100 1.61 6.55 8.69 10.99 11.4212.67 14.26 16.73 17.78 Sample A 0.94 2.54 3.19 3.70 4.01 3.96 5.16 7.068.86 Positive Control 1.29 21.15 32.72 42.30 47.07 53.73 64.53 83.1688.56 (SolkaFloc)

Example 30 Shake Flask Fermentation Using Pichia stipitis

Summary

Shake flask fermentation using Pichia stipitis was performed using fourcellulosic materials having the highest % performance from Table 36.

Protocol

Experiments were run under the parameters outlined in Tables 54-56.

TABLE 54 Equipment and Frequency of Maintenance Frequency of EquipmentManufacturer, Name Maintenance Shakers (2) B. Braun Biotech, CertomatBS-1 Quarterly Spectrophotometer Unicam, UV300 Biannual YSI BiochemInterscience, YSI Monthly Analyzer

TABLE 55 YSI Components Used in Shake Flask Study Component Reference#Lot # YSI Ethanol Membrane 2786 07M100361 YSI Ethanol Standard (3.2 g/L)2790 1271040 YSI Ethanol Buffer 2787 07J100215

TABLE 56 Chemicals Used for Shake Flask Fermentation ManufacturerReference# Lot# Urea ScholAR Chemistry 9472706 AD-7284-43 Yeast NitrogenBecton Dickinson 291940 7128171 Base Peptone Becton Dickinson 2116774303198 YM Broth Becton Dickinson 271120 6278265 Xylose Alfa AesarA10643 10130919 Glucose Fisher Scientific BP350-1 030064

Seed Development

For all the following shake flask experiments the seed flasks wereprepared using the following procedure.

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol were storedat −75° C. A portion of the thawed working cell bank material wasstreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for 2 days at 4° C. beforeuse. A 250 mL Erlenmeyer flask containing 100 mL of medium (40 g/Lglucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone,40 g/L xylose, pH 5.0) was inoculated with one colony and incubated for24 hours at 25° C. and 100 rpm. After 23 hours of growth, a sample wastaken and analyzed for optical density (600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, oneflask (called the Seed Flask) at an optical density of between 4 and 8and with a clean Gram stain was used to inoculate all of the testflasks.

Three experiments were run using samples A132-10, A132-100, G132-10, andG132-100. Experiment #1 tested these four samples for ethanolconcentration at varying concentrations of xylose and at constantconcentrations of glucose. Experiment #2 tested these four samples forethanol concentration at double the concentration of feedstock used inthe experiments of Table 36. Finally, experiment #3 tested these foursamples for ethanol concentration while varying both the xylose and theglucose concentrations, simultaneously.

Experiment #1—Varying the Xylose Concentration

Four cellulosic samples (A132-10, A132-100, G132-10, and G132-100) weretested at varying xylose concentrations as listed in Table 57 below.

TABLE 57 Media Composition of Experiment #1 Flasks Xylose ConcentrationGlucose Concentration Treatment (g/L) (g/L) 100% Xylose 40.0 40.0 50%Xylose 20.0 40.0 25% Xylose 10.0 40.0 10% Xylose 4.0 40.0 0% Xylose 0.040.0

The test vessels (a total of 40, 250 mL Erlenmeyer flasks) contained 100mL of medium. Five different types of media were prepared with theamount of xylose and glucose outlined in Table 57. In addition, themedia contained 1.7 g/L yeast nitrogen base (Becton Dickinson#291940)2.27 g/L urea (ScholAR Chemistry #9472706), and 6.56 g/L peptone (BectonDickinson #211677). All flasks were autoclaved empty at 121° C. and 15psi and filter sterilized (0.22) 1 m filter) media was added to theflasks prior to the addition of the test materials. Flasks were held atroom temperature for 4 days and inspected for contamination (cloudiness)prior to use. The test materials were not sterilized, as autoclavingwill change the content of the samples and filter sterilization notappropriate for sterilization of solids. The test samples (A132-10,A132-100, G132-10, and G132-100 at 5 g per 100 mL) were added at thetime of inoculation (rather than prior to) to reduce the possibility ofcontamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm for 72 hours.

Unfortunately, one flask (sample A132-100 with 100% Xylose) was brokenduring the testing. Therefore, all results past 24 hours of incubationare reported as a single flask. After 72 hours of incubation, 100% ofthe original amount of cellulosic material (5.0 g) was added to the 100%Xylose flasks (7 flasks in total, one flask containing sample A132-100was broken) and incubated as above for an additional 48 hours.

TABLE 58 Addition of Feedstock to 100% Xylose Flasks at Incubation Time27 hours Feedstock Added at 72 hours (grams) A132-10 5 A132-100 5G132-10 5 G132-100 5

Analysis

Samples were taken from the 40 test flasks at incubation times of 0, 6,12, 24, 36, 48, and 72 hours. In addition, samples were taken at 24 and48 hours post-addition of the second feedstock amount in the 100% Xyloseflasks (see Table 58).

A total of 292 samples were analyzed for ethanol concentration using aYSI Biochem Analyzer based on the alcohol dehydrogenase assay (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. Of note, time 0 samples requiredfiltration through a 0.45) 1 m syringe filter. The samples will bediluted to between 0-3.2 g/L ethanol prior to analysis. A standard of2.0 g/L ethanol was analyzed approximately every 30 samples to ensurethe integrity of the membrane was maintained.

A total of 47 samples were analyzed for cell count. Samples will betaken at 72 hours incubation and 48 hours post-addition of morecellulosic material. Appropriately diluted samples were mixed with 0.05%Trypan blue and loaded into a Neubauer haemocytometer. The cells werecounted under 40× magnification.

Experiment #2—Analysis of 2× Feedstock Concentration

The test vessels (a total of 8, 250 mL Erlenmeyer flasks) contained 100mL of medium. The media contained 40 g/L glucose, 40 g/L xylose, 1.7 giLyeast nitrogen base (Becton Dickinson#291940) 2.27 g/L urea (ScholARChemistry #9472706), and 6.56 g/L peptone (Becton Dickinson #211677).Flasks were prepared as in Experiment #1. The test samples (A132-10,A132-100, G132-10, and G132-100 at 10 g per 100 mL) were added at thetime of inoculation (rather than prior to) to reduce the possibility ofcontamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm above for 72 hours.

Analysis

Samples were from the 8 test flasks at an incubation time of 0, 6, 12,24, 36, 48, and 72 hours. Ethanol analysis of the 56 samples wereperformed as per experiment #1 and are reported in Table 59. A cellcount was performed on the 72 hour sample as per experiment #1 and ispresented in Table 60.

TABLE 59 Ethanol Concentration in Flasks with Double Feedstock SampleEthanol Concentration (g/L) Time A132-10 A132-100 G132-10 G132-100 01.38 0.26 0.12 0.11 6 1.75 0.21 0.20 0.10 12 2.16 0.73 0.69 0.31 2419.05 15.35 16.55 12.60 36 21.75 17.55 18.00 15.30 48 26.35 23.95 24.6520.65 72 26.95 27.35 28.90 27.40

TABLE 61 Media Composition of Experiment #3 Flasks Xylose ConcentrationGlucose Concentration Treatment (g/L) (g/L) 50% Sugar 20.0 20.0 25%Sugar 10.0 10.0 10% Sugar 4.0 4.0 0% Sugar 0.0 0Experiment #3—Varying Xylose and Glucose Concentrations

Four Cellulosic samples (A132-10, A132-100, G132-10, and G132-100) weretested at varying xylose and glucose concentrations as listed in thetable below (Table 60)

TABLE 60 Cell Concentration at 72 hour Incubation Time in Flasks withDouble Feedstock Sample Cell Concentration (×10⁸/ml) A132-10 4.06A132-100 5.37 G132-10 5.18 G132-100 4.47

TABLE 61 Media Composition of Experiment #3 Flasks Xylose ConcentrationGlucose Concentration Treatment (g/L) (g/L) 50% Sugar 20.0 20.0 25%Sugar 10.0 10.0 10% Sugar 4.0 4.0 0% Sugar 0.0 0

The test vessels (a total of 32, 250 mL Erlenmeyer flasks) contained 100mL of medium. Four different types of media were prepared with theamount of xylose and glucose outlined in Table 61. In addition, themedia contained 1.7 g/L yeast nitrogen base (Becton Dickinson#291940)2.27 g/L urea (ScholAR Chemistry #9472706), and 6.56 g/L peptone (BectonDickinson #211677). The flasks were prepared as per Experiment #1. Thetest samples (A132-10, A132-100, G132-10, and G132-100) were added atthe time of inoculation (rather than prior to) to reduce the possibilityof contamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm for 72 hours.

Analysis

Samples were taken from the 32 test flasks at an incubation time of 0,6, 12, 24, 36, 48, and 72 hours (see Tables 62-65). A total of 224samples were analyzed for ethanol concentration using the YSI BiochemAnalyzer based on the alcohol dehydrogenase assay (YSI, Interscience).Samples were centrifuged at 14,000 rpm for 20 minutes and thesupernatant stored at −20° C. Of note, some of the samples requiredcentrifugation and then filtration through a 0.45 lm syringe filter. Thesamples were diluted to between 0-3.2 g/L ethanol prior to analysis. Astandard of 2.0 g/L ethanol was analyzed approximately every 30 samplesto ensure the integrity of the YSI membrane was maintained.

TABLE 62 Ethanol Results Sample A132-10 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.43 0.42 0.42 0.41 0.390.53 0.57 0.56 0.56 6 1.16 1.16 1.15 1.16 1.12 0.93 0.91 0.83 0.88 121.72 1.86 1.71 1.79 1.90 1.21 2.13 2.47 2.32 24 15.55 15.90 17.05 17.0516.95 1.02 4.88 9.77 13.35 36 17.10 17.40 20.25 21.35 20.25 1.29 4.279.99 17.55 48 16.40 17.05 19.70 23.00 26.80 1.47 3.03 8.33 16.60 7215.15 15.55 19.25 21.85 28.00 1.14 1.52 5.08 14.20 24 hours — — — —23.15 — — — — post- addition 48 hours — — — — 21.55 — — — — post-addition *Analysis from experiment #3.

TABLE 63 Ethanol Results Sample A132-100 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.11 0.09 0.17 0.20 0.180.12 0.14 0.09 0.13 6 0.13 0.15 0.15 0.15 0.14 0.10 0.11 0.11 0.13 120.88 1.00 1.18 1.25 0.89 0.18 1.58 1.55 1.57 24 15.90 15.70 16.50 16.0514.60** 0.18 3.33 7.99 11.15 36 16.00 17.90 16.90 19.45 17.80** 0.212.85 8.37 16.10 48 15.75 16.70 19.30 22.15 27.00** 0.54 1.47 7.54 15.6072 14.85 15.35 18.55 21.30 28.50** 0.78 0.51 4.47 12.90 24 hours — — — —24.80** — — — — post- addition 48 hours — — — — 23.60** — — — — post-addition *Analysis from experiment #3. **All results based on analysisof one flask.

TABLE 64 Ethanol Results Sample G132-10 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.09 0.08 0.08 0.08 0.080.05 0.05 0.05 0.06 6 0.14 0.13 0.14 0.14 0.13 0.11 0.12 0.11 0.12 121.01 0.96 1.00 0.87 1.14 0.48 1.60 1.79 1.71 24 15.90 15.70 16.30 16.0514.60 0.13 3.96 8.54 11.10 36 15.10 17.45 16.80 18.75 22.15 0.09 3.028.69 16.55 48 15.95 16.90 19.25 21.10 24.00 0.07 2.05 8.10 16.50 7213.50 15.80 18.55 21.25 26.55 0.09 0.11 5.55 14.15 24 hours — — — —24.95 — — — — post- addition 48 hours — — — — 24.20 — — — — post-addition *Analysis from experiment #3.

TABLE 65 Ethanol Results Sample G132-100 Ethanol Concentration (g/L)Sample 0% 10% w/v 25% w/v 50% w/v 100% w/v 0% 10% 25% 50% Time XyloseXylose Xylose Xylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.04 0.040.04 0.04 0.05 0.05 0.05 0.05 0.06 6 0.07 0.07 0.08 0.08 0.07 0.04 0.050.05 0.06 12 0.60 0.56 0.67 0.58 0.71 0.13 1.37 1.48 1.44 24 13.05 14.4514.90 13.95 12.05 0.03 3.67 7.62 10.55 36 15.10 17.10 18.25 18.20 19.250.01 3.09 8.73 16.10 48 14.40 17.00 19.35 22.55 24.45 0.01 1.91 7.7615.85 72 14.70 15.40 18.45 22.10 27.55 0.03 0.01 5.08 14.30 24 hours — —— — 25.20 — — — — post- addition 48 hours — — — — 24.60 — — — — post-addition *Analysis from experiment #3.

Samples were taken at 72 hours incubation for cell counts (see Tables66-67). Appropriately diluted samples were mixed with 0.05% Trypan blueand loaded into a Neubauer haemocytometer. The cells were counted under40× magnification.

Results

One seed flask was used to inoculate all Experiment #1 and #2 test flaskoptical density (600 nm) of the seed flask was measured to be 5.14 andthe cell concentration was 4.65×108 cells/mL (Tables 65-66). Therefore,the initial concentration of cells in the test flasks was approximately4.65×106 cells/mL.

A second seed flask was used to inoculate Experiment #3 flasks. Theoptical density (600 nm) of the seed flask was 5.78 and the cellconcentration was 3.75×108 cells/mL. Therefore, the initialconcentration of cells in the test flasks was approximately 3.75×106cells/mL.

TABLE 66 Cell Counts at Incubation Time of 72 hours Cell Concentration(×10⁸/ml) 0% 10% 25% 50% 100% 0% 10% 25% 50% Sample Xylose Xylose XyloseXylose Xylose Sugar Sugar Sugar Sugar A132-10 0.37 0.63 3.72 4.92 4.050.26 0.22 0.26 1.54 A132-100 0.99 1.07 0.99 0.78 1.97 0.03* 0.33 0.441.81 G132-10 0.95 4.50 2.67 2.67 3.82 0.01* 0.17 0.49 1.92 G132-100 6.534.02 4.84 4.47 5.29 0.01* 0.33 0.89 2.22 *Samples were heavilycontaminated after 72 hours of growth. This is expected because thePichia did not grow well without sugar added, and contaminants (from thenon-sterile samples) were able to out-grow the Pichia.

TABLE 67 Cell Counts at Incubation Time of 48 hours Post-Addition (100%Xylose and Glucose) Sample Cell Concentration (×10⁸/ml) A132-10 10.17A132-100 3.38 G132-10 3.94 G132-100 6.53

Example 31 Toxicity Testing of Lignocellulosic Samples Against P.stipitis and S. cerevisiae

Summary

Thirty-seven samples were analyzed for toxicity against twoethanol-producing cultures, Saccharomyces cerevesiae and Pichiastipitis. In this study, glucose was added to the samples in order todistinguish between starvation of the cultures and toxicity of thesamples.

TABLE 68 Conditions for Toxicity Testing Organism Saccharomycescerevisiae Pichia stipitis Variable ATCC 24858 NRRL Y-7124 Inoculation0.5-1 1 Volume (ml) (target 6-7 × 10⁵ cells/ml) (target 3-4 × 10⁶cells/ml) Test Repetition Single Flasks Incubation Tem- 25° C. 25° C.perature (±1° c.) Shaker Speed 200 125 (rpm) Type of Container 500 mlErlenmeyer Flask 250 ml Erlenmeyer Flask Media volume 100 ml 100 mlTotal Incubation  72  72 time (hours) Ethanol Analysis 0, 6, 12, 24, 36,48, 72 0, 6, 12, 24, 36, 48, 72 (hours) Cell Counts 24, 72 24, 72(hours) pH 0 hours 0 hours

Protocol

A summary of the protocol used is listed in Table 68. A description ofthe chemicals used in toxicity testing is listed in Table 69. Twocontrol flasks (no sample added) were performed for each microorganismfor each week of testing. A total of 82 flasks were analyzed.

During the experiments, no ethanol or cells appeared in the P. stipitisflasks containing samples C, C-1e, C-5e, and C-10e in the first 24 hoursof incubation. In order to confirm the results, the test was repeated.The second test confirmed some inhibition of P. stipitis growth whensamples C, CIE, C5E, and CIOE were added to the flasks.

TABLE 69 Chemicals and Materials Used for Toxicity Testing MediaComponent Manufacturer Reference# Lot# Urea ScholAR Chemistry 9472706AD-7284-43 Yeast Nitrogen Becton Dickinson 291940 7128171 Base PeptoneBecton Dickinson 211677 4303198 Xylose Alfa Aesar A10643 10130919Glucose Sigma G-5400 107H0245 Yeast Extract Becton Dickinson 2886204026828 YM Broth Becton Dickinson 271120 6278265

TABLE 70 YSI Components Used in Toxicity Study Component Catalogue # YSIEthanol Membrane 2786 YSI Ethanol Standard (3.2 g/L) 2790 YSI EthanolBuffer 2787

Test Samples

Seven test samples (all with the C designation) were ground using acoffee grinder suitable for small samples. The samples were ground to aconsistent particle size (between samples) with the naked eye. Samplenumber C-100e ground easily to a small particle size.

All samples were added to the flasks at a concentration of 50 grams perliter with the exception of the six P samples (25 grams per liter).These samples were white to off-white in color and visually fluffy andthe flasks would not mix properly (not enough free liquid) at the 50grams per liter concentration. Samples S dissolved easily and could inthe future be added to the flasks at a higher concentration. Samples Aand G could be added at 100 grams per liter in the future.

Testing was performed using the two microorganisms as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

A working cell bank of S. cerevisiae ATCC 24858 was prepared from arehydrated lyophilized culture obtained from American Type CultureCollection. Cryovials containing S. cerevisiae culture in 15% v/vglycerol are stored at −75° C. A portion of the thawed working cell bankmaterial will be streaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH5.0) and incubated at 30° C. for 2 days. A 250 mL Erlenmeyer flaskcontaining 50 mL of medium (20 g/L glucose, 3 g/L yeast extract, and 5.0g/L peptone, pH 5.0) was inoculated with one colony from the YM plateand incubated for 24 hours at 25° C. and 200 rpm. After 23 hours ofgrowth, a sample was taken and analyzed for optical density (600 nm in aUV spectrophotometer) and purity (Gram stain). Based on these results,one flask (called the Seed Flask) with an OD of 9-15 and pure Gram stainwas to be used for inoculating the growth flasks. After 23 hours ofgrowth, the seed flask had a low OD (5.14) and cell count (1.35×108cells/mL). Of note, the colony taken from the seed plate was smallerthan usual. Therefore, 0.5 mL of seed material (as opposed to theplanned 0.1 mL) was added to each test vessel.

The test vessels were 500 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved at 121° C.and 15 psi prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving would change the contentof the samples. The test samples were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 0.5-1.0 mL (0.5-1.0% v/v) of seed flaskmaterial was added to each flask. The flasks were incubated as describedabove for 72 hours.

Pichia stipitis (ARS Culture Collection)

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol are storedat −75° C. A portion of the thawed working cell bank material wasstreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for up to 5 days at 4° C.before use. A 250 mL Erlenmeyer flask containing 100 mL of medium (40g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/Lpeptone, 40 g/L xylose, pH 5.0) was inoculated with one colony andincubated for 24 hours at 25° C. and 125 rpm. After 23 hours growth, asample was taken and analyzed for optical density (600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, oneflask (called the Seed Flask) at an optical density of 5-9 and with apure Gram Stain was used to inoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved empty at 121°C. and 15 psi and filter sterilized (0.22) l·m filter) medium added tothe flasks prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving would change the contentof the samples and filter sterilization not appropriate forsterilization of solids. The test samples were added at the time ofinoculation (rather than prior to) to reduce the possibility ofcontamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated asdescribed above for 72 hours.

Analysis

Samples were taken from seed flasks just prior to inoculation and eachtest flask at 24 and 72 hours and analyzed for cell concentration usingdirect counts. Appropriately diluted samples of S. cerevisiae and P.stipitis were mixed with 0.05% Trypan blue, loaded into a Neubauerhaemocytometer. The cells were counted under 40× magnification.

Samples were taken from each flask at 0, 6, 12, 24, 36, 48 and 72 hoursand analyzed for ethanol concentration using the YSI Biochem Analyzerbased on the alcohol dehydrogenase assay (YSI, Interscience). Sampleswere centrifuged at 14,000 rpm for 20 minutes and the supernatant storedat −20° C. The samples will be diluted to 0-3.2 g/L ethanol prior toanalysis. A standard of 2.0 g/L ethanol was analyzed approximately every30 samples to ensure the integrity of the membrane was maintained duringanalysis.

Calculations

The following calculations were used to compare the cell counts andethanol concentration to the control flasks.% performance=(concentration of ethanol in test flask/ethanol incontrol)*100%cells=(number of cells in test flask/number of cells in controlflask)*100

Results

The S. cerevisiae seed flask had an optical density (600 nm) of 5.14 anda cell concentration of 1.35×10⁸ cells/mL. One half mL of seed flaskmaterial was added to each of the test flasks. Therefore, the startingcell concentration in each flask was 6.75×10⁵/mL. During the second weekof testing, the S. cerevisiae seed flask had an optical density (600 nm)of 4.87 and a cell concentration of 3.15×10⁷ cells/mL. One mL of seedflask material was added to each of the test flasks. Therefore, thestarting cell concentration in each flask was 6.30×10⁵/mL. The pH of theS. cerevisiae flasks at a sample time of 0 hours is presented in Table71. The pH of the flask contents was within the optimal pH for S.cerevisiae growth (pH 4-6). No pH adjustment was required.

TABLE 71 pH of S. cerevisiae flasks at sample time 0 hours Sample SampleNumber pH Number pH p 5.04 c 5.46 P1E 4.99 C1E 5.54 P5E 5.04 C5E 5.50P10E 4.98 C10E 5.33 P50E 4.67 C30E 5.12 P100E 4.43 C50E 4.90 G 5.45C100E 4.66 G1E 5.47 ST 5.11 G5E 5.46 ST 1E 5.06 G10E 5.39 ST 5E 4.96G50E 5.07 ST10E 4.94 A 5.72 ST30E 5.68 A1E 5.69 ST50E 4.48 A5E 5.62ST100E 4.23 A10E 5.61 control A 5.02 A50E 5.74 control B 5.04 S* 5.10S1E 5.08 S5E 5.07 S10E 5.04 S30E 4.84 S50E 4.57 S100E 4.33 *“S” refersto sucrose *“C” refers to com *“ST” refers to starch

The ethanol concentration and performance in the S. cerevisiae flasksare presented in Table 72 and 73. The highest ethanol concentrationswere produced by the S series.

TABLE 72 Ethanol Concentration in S. cerevisiae flasks Sample EthanolConcentration {g/L) at the following times (hours) Number 0 6 12 24 3648 72 p 0.0 0.04 0.38 5.87 7.86 5.41 1.04 PIE 0.0 0.03 0.28 5.10 8.035.46 0.58 P5E 0.0 0.04 0.57 8.84 6.38 3.40 0.04 P10E 0.0 0.05 0.65 6.637.66 5.57 1.40 P50E 0.0 0.03 0.26 2.80 5.85 8.59 5.68 P100E 0.0 0.020.12 3.64 8.26 7.51 3.03 G 0.0 0.04 0.57 10.20 8.24 6.66 2.84 G1E 0.00.05 0.46 10.20 9.24 6.94 2.84 G5E 0.1 0.11 0.44 10.00 8.7 6.36 0.88G10E 0.0 0.04 0.40 9.97 8.41 5.79 0.11 G50E 0.0 0.05 0.48 9.72 8.33 6.132.38 A 0.2 0.38 0.48 8.43 8.76 7.09 4.66 A1E 0.3 0.44 0.79 9.66 8.9 7.182.64 A5E 0.5 0.45 0.99 9.44 8.96 7.56 3.80 A10E 0.5 0.55 0.93 9.58 8.336.28 1.40 A50E 0.2 0.08 0.38 9.38 8.01 5.99 0.98 S 0.0 0.03 0.39 5.737.06 10.10 15.90 S1E 0.0 0.06 0.31 7.24 9.52 12.10 14.90 S5E 0.0 0.050.34 5.87 7.68 11.90 19.00 S10E 0.0 0.04 0.35 5.88 7.72 11.50 19.30 S30E0.0 0.05 0.09 5.94 7.97 11.20 20.40 S50E* 0.1 0.19 0.47 5.46 7.96 13.0018.30 S100E 0.1 0.10 0.21 7.00 10.6 13.80 12.70 C 0.0 0.04 0.32 8.477.57 5.48 6.40 C1E 0.0 0.06 0.37 8.93 7.86 5.99 1.37 C5E 0.0 0.05 0.489.32 7.92 5.69 1.41 C10E 0.0 0.04 0.52 9.14 7.67 5.34 0.35 C30E 0.0 0.050.28 9.15 8.15 5.84 2.47 C50E 0.0 0.06 0.44 9.31 7.79 5.78 1.79 C100E0.0 0.06 0.58 9.06 6.85 5.95 1.09 ST 0.0 0.05 0.99 8.54 6.69 5.09 0.42ST1E 0.0 0.04 0.70 8.87 7.29 4.81 1.04 ST5E 0.0 0.04 0.52 8.61 7.16 4.970.85 ST10E 0.0 0.05 0.33 8.97 7.05 5.26 0.68 ST30E 0.0 0.04 0.71 8.476.96 4.89 0.21 ST50E 0.0 0.07 0.34 8.46 8.19 7.04 3.20 ST100E 0.0 0.100.30 9.30 8.62 7.29 4.23 control A 0.0 0.07 0.85 5.92 8.18 7.81 6.26control B 0.0 0.04 0.27 4.86 6.43 8.01 6.75 control A* 0.0 0.21 1.365.19 7.31 7.55 5.16 control B* 0.0 0.20 1.18 5.16 5.96 7.62 5.32*analyzed week 2 See Table 72 for Sample Number key

TABLE 73 Performance in S. cerevisiae flasks Sample Performance (%) atthe following times (hours) Number 24 36 48 72 p 108.9 107.6 68.4 16.0P1E 94.6 109.9 69.0 8.9 P5E 164.0 87.3 43.0 0.6 P10E 123.0 104.9 70.421.5 P50E 51.9 80.1 108.6 87.3 P100E 67.5 113.1 94.9 46.5 G 189.2 112.884.2 43.6 G1E 189.2 126.5 87.7 43.6 G5E 185.5 119.1 80.4 13.5 G10E 185.0115.1 73.2 1.7 G50E 180.3 114.0 77.5 36.6 A 156.4 119.9 89.6 71.6 A1E179.2 121.8 90.8 40.6 A5E 175.1 122.7 95.6 58.4 A10E 177.7 114.0 79.421.5 A50E 174.0 109.7 75.7 15.1 S 106.3 96.6 127.7 244.2 S1E 134.3 130.3153.0 228.9 S5E 108.9 105.1 150.4 291.9 S10E 109.1 105.7 145.4 296.5S30E 110.2 109.1 141.6 313.4 S50E* 105.5 119.9 171.3 349.2 S100E 129.9145.1 174.5 195.1 C 157.1 103.6 69.3 98.3 C1E 165.7 107.6 75.7 21.0 C5E172.9 108.4 71.9 21.7 C10E 169.6 105.0 67.5 5.4 C30E 169.8 111.6 73.837.9 C50E 172.7 106.6 73.1 27.5 C100E 168.1 93.8 75.2 16.7 ST 158.4 91.664.3 6.5 ST1E 164.6 99.8 60.8 16.0 ST5E 159.7 98.0 62.8 13.1 ST10E 166.496.5 66.5 10.4 ST30E 157.1 95.3 61.8 3.2 ST50E 157.0 112.1 89.0 49.2ST100E 172.5 118.0 92.2 65.0 control A 109.8 112.0 98.7 96.2 control B90.2 88.0 101.3 103.7 control A* 100.3 110.1 99.5 98.5 control B* 99.799.7 100.4 101.5 *analyzed week 2

The cell concentration and % cells in the S. cerevisiae flasks werepresented in Table 74. High cell counts were observed in all flasks;however, not all of the cells appear to be making ethanol.

TABLE 74 S. cerevisea Cell Counts and % Cells Cell Count % Cells Sample(Cells × 10⁸) (Count/Count control) × 100 Number 24 hours 72 hours 24hours 72 hour P 0.6 0.96 97.7 139.0 P1E 0.3 1.18 54.1 170.9 P5E 1.1 1.93177. 279.5 P50E 0.3 1.40 49.4 202.8 P100E 0.4 1.94 70.6 281.0 G 0.7 3.48116.5 504.0 G1E 0.6 3.65 107.1 528.6 G5E 0.6 3.87 96.5 560.5 G10E 0.72.73 109.5 395.4 G50E 0.4 2.10 71.8 304.1 A 0.5 3.53 86.0 511.2 A1E 0.83.45 130.7 499.6 A5E 0.6 3.53 104.8 511.2 A10E 0.5 1.95 83.6 282.4 A50E0.6 1.62 103.5 234.6 S 0.4 1.11 69.5 160.8 S1E 0.4 1.10 68.2 159.3 S5E0.2 0.99 36.5 143.4 S10E 0.3 0.73 61.2 105.4 S30E 0.3 0.71 48.3 102.1S50E* 0.4 0.90 86.5 196.5 S100E 0.5 0.84 82.4 121.7 C 0.4 1.81 70.6262.1 C1E 0.7 2.40 110.6 347.6 C5E 0.5 2.33 83.6 337.4 C10E 0.7 1.55120.0 224.5 C30E 0.7 1.80 117.6 260.7 C50E 0.6 1.70 100.1 246.2 C100E0.8 1.51 127.1 218.7 ST 0.7 1.75 117.6 253.4 ST1E 0.5 1.36 89.4 197.0ST5E 0.5 1.49 90.7 215.8 ST10E 0.6 1.32 95.4 191.2 ST30E 0.5 0.60 91.886.9 ST50E 0.5 1.30 91.8 188.3 ST100E 0.4 1.24 63.5 179.6 control A 0.80.79 127.1 114.1 control B 0.4 0.59 72.9 85.9 control A* 0.6 0.42 131.291.7 control B* 0.3 0.50 69.0 108.1

The P. stipitis seed flask had an optical density (600 nm) of 5.01 and acell concentration of 3.30×10⁸ cells/mL. One mL of seed flask materialwas added to each of the test flasks. Therefore, the starting cellconcentration in each flask was 3.30×10⁶/mL. During the second week oftesting, the P. stipitis seed flask had an optical density (600 nm) of5.45 and a cell concentration of 3.83×10⁸ cells/mL. One mL of seed flaskmaterial was added to each of the test flasks. Therefore, the startingcell concentration in each flask was 3.83×10⁶/mL. The pH of the P.stipitis flasks at a sample time of 0 hours is presented in Table 75.The pH of the flask contents was within the optimal pH for P. stipitisgrowth (pH 4-7). No pH adjustment was required.

TABLE 75 pH of P. stipitis Flasks at Sample Time 0 Hours Sample SampleNumber pH Number pH p 4.91 C 5.36 P1E 4.87 C1E 5.30 P5E 4.90 C5E 5.29P10E 4.78 C10E 5.06 P50E 4.46 C30E 4.89 P100E 4.24 C50E 4.70 G 5.45C100E 4.59 G1E 5.43 ST 4.93 G5E 5.48 ST1E 4.90 G10E 5.32 ST5E 4.81 G50E4.99 ST10E 4.83 A 5.69 ST30E 4.91 A1E 5.66 ST50E 4.24 A5E 5.60 ST100E4.07 A10E 5.58 control A 4.93 A50E 5.69 control B 4.91 S 5.00 S1E 4.94S5E 4.86 S10E 4.78 S30E 4.51 S50E 4.27 S100E 4.08

The ethanol concentration and performance in the P. stipitis flasks arepresented in Table 76 and 77. The highest ethanol concentrations werethe G and A series. Flasks C-30e, C-50e, and C-100e also contained highconcentrations of ethanol. The cell concentration and % cells in the P.stipitis flasks are presented in Table 78. Low cell concentrations wereobserved in the flasks with the S designations. Low cell counts werealso observed in flasks containing samples C, CIE, C5E, and CI0E at the24 hour sample time.

TABLE 76 Ethanol concentration in P. stipitis flasks Sample EthanolConcentration (g/L) at the following times (hours) Number 0 6 12 24 3648 72 P 0.01 0.05 0.26 4.98 8057 14.10 17.00 P1E 0.02 0.03 0.04 4.249.03 12.40 17.30 P5E 0.02 0.03 0.42 6.72 12.40 15.60 18.60 P10E 0.020.02 0.01 1.38 8.69 13.00 17.00 P50E 0.01 0.02 0.02 0.03 3.77 10.5016.90 P100E 0.02 0.03 0.02 3.75 10.50 15.60 18.80 G 0.02 0.08 0.20 10.8017.70 19.40 25.40 G1E 0.04 0.12 0.50 12.20 19.60 23.80 28.60 G5E 0.070.14 0.73 12.50 19.10 24.50 27.50 G10E 0.04 0.19 0.42 10.20 19.10 22.9028.20 G50E 0.05 0.22 0.25 8.73 18.40 22.20 28.00 A 0.13 0.28 0.82 16.1019.40 19.30 18.60 A1E 0.22 0.59 1.08 16.10 22.40 27.60 27.70 A5E 0.320.43 0.43 10.60 22.10 27.10 28.10 A10E 0.33 0.61 1.15 14.90 22.00 27.1027.90 A50E 0.30 0.10 0.47 13.40 20.20 24.80 27.10 S 0.01 0.01 0.26 3.687.50 10.20 13.30 S1E 0.02 0.02 0.22 4.98 9.22 11.60 14.20 S5E 0.02 0.020.19 4.25 8.50 11.70 14.70 S10E 0.03 0.02 0.17 2.98 8.87 11.90 14.70S30E 0.08 0.05 0.03 2.96 8.73 12.60 16.50 S50E 0.08 0.05 0.04 2.24 6.137.95 12.50 S100E 0.11 0.10 0.08 3.36 7.82 10.50 13.90 C* 0.02 0.03 0.050.23 1.66 2.68 6.57 C1E* 0.03 0.03 0.03 0.07 0.95 1.85 10.20 C5E* 0.030.02 0.04 0.05 0.37 1.59 4.80 C10E* 0.03 0.04 0.04 0.05 3.91 15.20 28.30C30E 0.01 0.03 0.60 12.30 21.20 26.00 27.20 C50E 0.02 0.02 0.45 12.3019.50 23.80 29.20 C100E 0.05 0.04 0.38 11.40 18.70 22.90 27.70 ST 0.030.03 0.37 6.69 10.70 13.50 10.90 ST1E 0.01 0.00 0.48 5.24 9.37 12.5015.70 ST5E 0.02 0.03 0.29 5.45 10.10 11.90 14.70 ST10E 0.02 0.02 0.425.60 9.44 12.20 14.90 ST30E 0.05 0.04 0.73 5.70 9.50 12.10 15.20 ST50E0.02 0.05 0.19 5.16 9.47 12.70 15.20 ST100E* 0.07 0.15 0.11 4.98 10.7015.40 18.80 control A 0.02 0.03 0.37 4.05 7.50 9.24 11.50 control B 0.020.02 0.30 4.22 7.44 9.44 11.50 control A* 0.02 0.05 0.69 4.86 8.69 11.1016.40 control B* 0.02 0.05 0.74 5.96 10.80 13.00 14.00 *analyzed week 2

TABLE 77 Performance in P. stipitis flasks Sample Performance (%) at thefollowing times (hours) Number 24 36 48 72 p 120.3 114.7 151.0 147.8 P1E102.4 120.9 132.8 150.4 P5E 162.3 166.0 167.0 161.7 P10E 33.3 116.3139.2 147.8 P50E 0.7 50.5 112.4 147.0 P100E 90.6 140.6 167.0 163.5 G260.9 236.9 207.7 220.9 G1E 294.7 262.4 254.8 248.7 G5E 301.9 255.7262.3 239.1 G10E 246.4 255.7 245.2 245.2 G50E 210.9 246.3 237.7 243.5 A388.9 259.7 206.6 161.7 A1E 388.9 299.9 295.5 240.9 A5E 256.0 295.9290.1 244.3 A10E 359.9 294.5 290.1 242.6 A50E 323.7 270.4 265.5 235.7 S88.9 100.4 109.2 115.7 S1E 120.3 123.4 124.2 123.5 S5E 102.7 113.8 125.3127.8 S10E 72.0 118.7 127.4 127.8 S30E 71.5 116.9 134.9 143.5 S50E 54.182.1 85.1 108.7 S100E 81.2 104.7 112.4 120.9 C* 4.2 17.0 22.2 43.2 C1E*1.4 9.7 15.4 67.1 C5E* 0.9 3.8 13.2 31.6 C10E* 0.9 40.1 126.1 246.1 C30E297.1 283.8 278.4 236.5 C50E 297.1 261.0 254.8 253.9 C100E 275.4 250.3245.2 240.9 ST 161.6 143.2 144.5 94.8 ST1E 126.6 125.4 133.8 136.5 ST5E131.6 135.2 127.4 127.8 ST10E 135.3 126.4 130.6 129.6 ST30E 137.7 127.2129.6 132.2 ST50E 124.6 126.8 136.0 132.2 ST100E* 120.3 109.7 127.8123.7 control A 97.8 100.4 98.9 100.0 control B 101.9 99.6 101.1 100.0control A* 89.8 89.1 92.1 107.9 control B* 110.2 110.8 107.9 92.1*analyzed in week 2

TABLE 78 P. stipitis Cell Counts and % Cells Cell Count % Cells Sample(Cells × 10⁸) (count/count control) × 100 Number 24 hours 72 hours 24hours 72 hours P 2.78 11.0 80.6 148.0 P1E 2.10 7.20 60.9 96.9 P5E 2.939.68 84.9 130.3 P10E 1.42 7.73 41.2 104.0 P50E 0.33 8.63 9.6 116.2 P100E1.58 8.25 45.8 111.0 G 1.50 14.2 43.5 191.1 G1E 3.90 8.10 113.0 109.0G5F 2.93 6.45 84.9 86.8 G10E 4.35 13.30 126.1 179.0 G50E 3.75 11.60108.7 156.1 A 7.43 8.55 215.4 115.1 A1E 4.13 9.53 119.7 128.3 A5E 3.689.75 106.7 131.2 A10E 4.50 7.50 130.4 100.9 A50E 6.23 5.33 180.6 71.7 S3.53 5.55 102.3 74.7 S1E 3.00 3.30 87.0 44.4 S5E 3.68 3.00 106.7 40.4S10E 1.73 5.78 50.1 77.8 S30E 2.55 5.48 73.9 73.8 S50E 2.63 6.15 76.282.8 S100E 2.25 4.43 65.2 59.6 C* 0.00 0.26 0.00 7.2 C1E* 0.00 0.36 0.009.9 C5E* 0.00 0.08 0.00 2.1 C10E* 0.00 5.85 0.00 160.7 C30E 5.78 4.20167.5 56.5 C50E 3.40 7.35 98.6 98.9 C100E 1.98 6.60 57.4 88.8 ST 2.557.65 73.9 103.0 ST1E 2.00 8.70 58.0 117.1 ST5E 1.85 6.75 53.6 90.8 ST10E1.83 5.40 53.0 72.7 ST30E 2.78 6.15 80.6 82.8 ST50E 1.33 3.45 38.6 46.4ST100E* 4.35 3.83 59.8 105.2 control A 3.60 7.13 104.3 96.0 control B3.30 7.73 95.7 104.0 control A* 7.50 3.23 103.0 88.7 control B* 7.054.05 96.8 111.3 *analyzed week 2

Cell Toxicity Results Summary

Zymomonas mobilis

As shown in FIG. 40A, elevated cell numbers (e.g., greater than thecontrol) were observed in samples containing P-132-10, G-132-10, andWS-132-10 at the 24 hour time point. Cell numbers in the presence of allother samples were comparable to the control. This observation indicatesthat the substrates were not toxic towards Z. mobilis for up to 24 hoursafter seeding.

At the 36 hour time point, a decrease in cell numbers (e.g., due to aloss of cells or cell death) was observed for all samples, including thecontrol. The greatest decrease in cell numbers was observed for thosesamples containing P-132-10, G-132-10. The likely cause of this effectis common to all samples, including the control. Thus, the cause of thiseffect is not the test substrates, as these vary in each sample, and arenot present in the control. Possible explanations for this observationinclude inappropriate culture conditions (e.g., temperature, mediacompositions), or ethanol concentrations in the sample.

As shown in FIG. 40B, all cells produced comparable amounts of ethanol(e.g., 5-10 giL) at each time point, irrespective of the substrate.Consistent with the cell number data presented in FIG. 40A, ethanolconcentration in each sample peaked at the 24 hour time point. Incontrast to the cell number data, ethanol concentration did not decreaseat subsequent time points. This was expected as ethanol was not removedfrom the system. In addition, this data suggests that ethanol productionin these samples may have resulted from fermentation of glucose in theculture media. None of the substrates tested appeared to increaseethanol production.

Together, FIGS. 40A and 40B suggest that ethanol concentrations aboveabout 6 g/L may be toxic to Z. mobilis. This data is also presented as apercentage normalized against the control, as shown in FIG. 40C.

Pichia stipitis

As shown in Chart 2A FIG. 41A, cell numbers were comparable to thecontrol. Furthermore, although slightly reduced cell numbers werepresent in samples containing G-132 and WS-132, reduced cell numberswere not observed for G-132-10, G-132-100, A-132-10, or A-132-100. Thus,it is unlikely that substrates G or A are toxic. Rather, the reducedcell numbers observed for G-132 and WS-132 are likely to have beencaused by an experimental anomaly or by the presence of unprocessedsubstrate somehow impeding cell growth. Overall, this data suggests thatglucose present in the control and experimental samples is likely to besufficient to promote optimal P. stipitis growth, and that the presenceof an additional substrate in the sample does not increase this growthrate. These results also suggest that none of the samples are toxic inP. stipitis.

As shown in FIG. 41B, despite the similar cell numbers reported in FIG.41B, greatly increased ethanol production was observed in all samplescontaining an experimental substrate. Ethanol concentrations increasedover time for each of the three time points tested. The highestconcentration of ethanol was observed for A-132-10 at the 48 hour timepoint (e.g., approximately 26.0 g/L). By comparing the substrateconcentrations with the highest levels of ethanol production with thecell number data presented in FIG. 41B, it can be seen that P. stipitisdo not appear to be sensitive to increasing ethanol concentrations.Furthermore, ethanol production does not appear to be related to cellnumber, but rather appears to be related to the type of substratepresent in the sample.

Together, the results presented in FIGS. 41A and 41B suggest that theexperimental substrates do not promote increased P. stipitis growth,however, they greatly increase the amount of ethanol produced by thiscell type. This data is also presented as a percentage normalizedagainst the control, as shown in FIG. 41C.

Saccharomyces cerevisiae

As shown in FIG. 42A, G-132-100, A-132, A-132-10, A-132-100, and WS-132promoted slightly elevated cell numbers compared to the control. Nosignificant reductions in cell number were observed for any sample.These results suggest that none of the samples are toxic in S.cerevisiae.

As shown in FIG. 42B, increased ethanol production was observed in cellstreated with each cell type compared to the control. Comparison of thosesamples containing the highest amount of ethanol with the cell numberdata presented in FIG. 42A suggests that ethanol concentrations inexcess of 5 g/L may have had an adverse effect on cell numbers. However,this observation is not the case for all samples.

This data is also presented as a percentage normalized against thecontrol, as shown in FIG. 42C.

In conclusion, none of the samples tested appeared to be toxic in Z.mobilis, P. stipitis, or S. cerevisiae. Furthermore, P. stipitisappeared to be the most efficient of the three cell types for producingethanol from the experimental substrates tested.

Example 32 Alcohol Production Using Irradiation-Sonication Pretreatment

The optimum size for biomass conversion plants is affected by factorsincluding economies of scale and the type and availability of biomassused as feedstock. Increasing plant size tends to increase economies ofscale associated with plant processes. However, increasing plant sizealso tends to increase the costs (e.g., transportation costs) per unitof biomass feedstock. Studies analyzing these factors suggest that theappropriate size for biomass conversion plants can range from 2000 to10,000 dried tons of biomass feedstock per day. The plant describedbelow is sized to process 2000 tons of dry biomass feedstock per day.

FIG. 43 shows a process schematic of a biomass conversion systemconfigured to process switchgrass. The feed preparation subsystemprocesses raw biomass feedstock to remove foreign objects and provideconsistently sized particles for further processing. The pretreatmentsubsystem changes the molecular structure (e.g., reduces the averagemolecular weight and the crystallinity) of the biomass feedstock byirradiating the biomass feedstock, mixing the irradiated the biomassfeedstock with water to form a slurry, and applying ultrasonic energy tothe slurry. The irradiation and sonication convert the cellulosic andlignocellulosic components of the biomass feedstock into fermentablematerials. The primary process subsystem ferments the glucose and otherlow weight sugars present after pretreatment to form alcohols.

Feed Preparation

The selected design feed rate for the plant is 2,000 dry tons per day ofswitchgrass biomass. The design feed is chopped and/or shearedswitchgrass.

Biomass feedstock, in the form of bales of switchgrass, is received bythe plant on truck trailers. As the trucks are received, they areweighed and unloaded by forklifts. Some bales are sent to on-sitestorage while others are taken directly to the conveyors. From there,the bales are conveyed to an automatic unwrapping system that cuts awaythe plastic wrapping and/or net surrounding the bales. The biomassfeedstock is then conveyed past a magnetic separator to remove trampmetal, after which it is introduced to shredder-shearer trains where thematerial is reduced in size. Finally, the biomass feedstock is conveyedto the pretreatment subsystem.

In some cases, the switchgrass bales are wrapped with plastic net toensure they don't break apart when handled, and may also be wrapped inplastic film to protect the bale from weather. The bales are eithersquare or round. The bales are received at the plant from off-sitestorage on large truck trailers.

Since switchgrass is only seasonally available, long-term storage isrequired to provide feed to the plant year-round. Long-term storage willlikely consist of 400-500 acres of uncovered piled rows of bales at alocation (or multiple locations) reasonably close to the ethanol plant.On-site short-term storage is provided equivalent to 72 hours ofproduction at an outside storage area. Bales and surrounding access waysas well as the transport conveyors will be on a concrete slab. Aconcrete slab is used because of the volume of traffic required todeliver the large amount of biomass feedstock required. A concrete slabwill minimize the amount of standing water in the storage area, as wellas reduce the biomass feedstock's exposure to dirt. The stored materialprovides a short-term supply for weekends, holidays, and when normaldirect delivery of material into the process is interrupted.

The bales are off-loaded by forklifts and are placed directly onto baletransport conveyors or in the short-term storage area. Bales are alsoreclaimed from short-term storage by forklifts and loaded onto the baletransport conveyors.

Bales travel to one of two bale unwrapping stations. Unwrapped bales arebroken up using a spreader bar and then discharged onto a conveyor,which passes a magnetic separator to remove metal prior to shredding. Atramp iron magnet is provided to catch stray magnetic metal and ascalping screen removes gross oversize and foreign material ahead ofmultiple shredder-shearer trains, which reduce the biomass feedstock tothe proper size for pretreatment. The shredder-shearer trains includeshredders and rotary knife cutters. The shredders reduce the size of theraw biomass feedstock and feed the resulting material to the rotaryknife cutters. The rotary knife cutters concurrently shear the biomassfeedstock and screen the resulting material.

Three storage silos are provided to limit overall system downtime due torequired maintenance on and/or breakdowns of feed preparation subsystemequipment. Each silo can hold approximately 55,000 cubic feet of biomassfeedstock (3 hours of plant operation).

Pretreatment

A conveyor belt carries the biomass feedstock from the feed preparationsubsystem 110 to the pretreatment subsystem 114. As shown in FIG. 44, inthe pretreatment subsystem 114, the biomass feedstock is irradiatedusing electron beam emitters, mixed with water to form a slurry, andsubjected to the application of ultrasonic energy. As discussed above,irradiation of the biomass feedstock changes the molecular structure(e.g., reduces the average molecular weight and the crystallinity) ofthe biomass feedstock. Mixing the irradiated biomass feedstock into aslurry and applying ultrasonic energy to the slurry further changes themolecular structure of the biomass feedstock. Application of theradiation and sonication in sequence may have synergistic effects inthat the combination of techniques appears to achieve greater changes tothe molecular structure (e.g., reduces the average molecular weight andthe crystallinity) than either technique can efficiently achieve on itsown. Without wishing to be bound by theory, in addition to reducing thepolymerization of the biomass feedstock by breaking intramolecular bondsbetween segments of cellulosic and lignocellulosic components of thebiomass feedstock, the irradiation may make the overall physicalstructure of the biomass feedstock more brittle. After the brittlebiomass feedstock is mixed into a slurry, the application of ultrasonicenergy further changes the molecular structure (e.g., reduces theaverage molecular weight and the crystallinity) and also can reduce thesize of biomass feedstock particles.

Electron Beam Irradiation

The conveyor belt 491 carrying the biomass feedstock into thepretreatment subsystem distributes the biomass feedstock into multiplefeed streams (e.g., 50 feed streams) each leading to separate electronbeam emitters 492. In this embodiment, the biomass feedstock isirradiated while it is dry. Each feed stream is carried on a separateconveyor belt to an associated electron beam emitter. Each irradiationfeed conveyor belt can be approximately one meter wide. Before reachingthe electron beam emitter, a localized vibration is induced in eachconveyor belt to evenly distribute the dry biomass feedstock over thecross-sectional width of the conveyor belt.

Electron beam emitter 492 (e.g., electron beam irradiation devicescommercially available from Titan Corporation, San Diego, Calif.) areconfigured to apply a 100 kilo-Gray dose of electrons applied at a powerof 300 kW. The electron beam emitters are scanning beam devices with asweep width of 1 meter to correspond to the width of the conveyor belt.In some embodiments, electron beam emitters with large, fixed beamwidths are used. Factors including belt/beam width, desired dose,biomass feedstock density, and power applied govern the number ofelectron beam emitters required for the plant to process 2,000 tons perday of dry feed.

Sonication

The irradiated biomass feedstock is mixed with water to form a slurrybefore ultrasonic energy is applied. There can be a separate sonicationsystem associated with each electron beam feed stream or severalelectron beam streams can be aggregated as feed for a single sonicationsystem.

In each sonication system, the irradiated biomass feedstock is fed intoa reservoir 1214 through a first intake 1232 and water is fed into thereservoir 1214 through second intake 1234. Appropriate valves (manual orautomated) control the flow of biomass feedstock and the flow of waterto produce a desired ratio of biomass feedstock to water (e.g., 10%cellulosic material, weight by volume). Each reservoir 1214 includes amixer 1240 to agitate the contents of volume 1236 and disperse biomassfeedstock throughout the water.

In each sonication system, the slurry is pumped (e.g., using a recessedimpeller vortex pump 1218) from reservoir 1214 to and through a flowcell 1224 including an ultrasonic transducer 1226. In some embodiments,pump 1218 is configured to agitate the slurry 1216 such that the mixtureof biomass feedstock and water is substantially uniform at inlet 1220 ofthe flow cell 1224. For example, the pump 1218 can agitate the slurry1216 to create a turbulent flow that persists throughout the pipingbetween the first pump and inlet 1220 of flow cell 1224.

Within the flow cell 1224, ultrasonic transducer 1226 transmitsultrasonic energy into slurry 1216 as the slurry flows through flow cell1224. Ultrasonic transducer 1226 converts electrical energy into highfrequency mechanical energy (e.g., ultrasonic energy), which is thendelivered to the slurry through booster 48. Ultrasonic transducers arecommercially available (e.g., from Hielscher USA, Inc. of Ringwood,N.J.) that are capable of delivering a continuous power of 16 kilowatts.

The ultrasonic energy traveling through booster 1248 in reactor volume1244 creates a series of compressions and rarefactions in process stream1216 with an intensity sufficient to create cavitation in process stream1216. Cavitation disaggregates components of the biomass feedstockincluding, for example, cellulosic and lignocellulosic materialdispersed in process stream 1216 (e.g., slurry). Cavitation alsoproduces free radicals in the water of process stream 1216 (e.g.,slurry). These free radicals act to further break down the cellulosicmaterial in process stream 1216. In general, about 250 MJ/m3 ofultrasonic energy is applied to process stream 1216 containing fragmentsof poplar chips. Other levels of ultrasonic energy (between about 5 andabout 4000 MJ/m3, e.g., 10, 25, 50, 100, 250, 500, 750, 1000, 2000, or3000) can be applied to other biomass feedstocks. After exposure toultrasonic energy in reactor volume 1244, process stream 1216 exits flowcell 24 through outlet 1222.

Flow cell 1224 also includes a heat exchanger 1246 in thermalcommunication with at least a portion of reactor volume 1244. Coolingfluid 1248 (e.g., water) flows into heat exchanger 1246 and absorbs heatgenerated when process stream 1216 (e.g., slurry) is sonicated inreactor volume 1244. In some embodiments, the flow of cooling fluid 1248into heat exchanger 1246 is controlled to maintain an approximatelyconstant temperature in reactor volume 1244. In addition or in thealternative, the temperature of cooling fluid 1248 flowing into heatexchanger 1246 is controlled to maintain an approximately constanttemperature in reactor volume 1244.

The outlet 1242 of flow cell 1224 is arranged near the bottom ofreservoir 1214 to induce a gravity feed of process stream 1216 (e.g.,slurry) out of reservoir 1214 towards the inlet of a second pump 1230which pumps process stream 1216 (e.g., slurry) towards the primaryprocess subsystem.

Sonication systems can include a single flow path (as described above)or multiple parallel flow paths each with an associated individualsonication unit. Multiple sonication units can also be arranged toseries to increase the amount of sonic energy applied to the slurry.

Primary Processes

A vacuum rotary drum type filter removes solids from the slurry beforefermentation. Liquid from the filter is pumped cooled prior to enteringthe fermentors. Filtered solids are passed to the post-processingsubsystem for further processing.

The fermentation tanks are large, low pressure, stainless steel vesselswith conical bottoms and slow speed agitators. Multiple first stagefermentation tanks can be arranged in series. The temperature in thefirst stage fermentation tanks is controlled to 30 degrees centigradeusing external heat exchangers. Yeast is added to the first stagefermentation tank at the head of each series of tanks and carriesthrough to the other tanks in the series.

Second stage fermentation consists of two continuous fermentors inseries. Both fermentors are continuously agitated with slow speedmechanical mixers. Temperature is controlled with chilled water inexternal exchangers with continuous recirculation. Recirculation pumpsare of the progressive cavity type because of the high solidsconcentration.

Off gas from the fermentation tanks and fermentors is combined andwashed in a counter-current water column before being vented to theatmosphere. The off gas is washed to recover ethanol rather than for airemissions control.

Post-Processing

Distillation

Distillation and molecular sieve adsorption are used to recover ethanolfrom the raw fermentation beer and produce 99.5% ethanol. Distillationis accomplished in two columns—the first, called the beer column,removes the dissolved C02 and most of the water, and the secondconcentrates the ethanol to a near azeotropic composition.

All the water from the nearly azeotropic mixture is removed by vaporphase molecular sieve adsorption. Regeneration of the adsorption columnsrequires that an ethanol water mixture be recycled to distillation forrecovery.

Fermentation vents (containing mostly C02, but also some ethanol) aswell as the beer column vent are scrubbed in a water scrubber,recovering nearly all of the ethanol. The scrubber effluent is fed tothe first distillation column along with the fermentation beer.

The bottoms from the first distillation contain all the unconvertedinsoluble and dissolved solids. The insoluble solids are dewatered by apressure filter and sent to a combustor. The liquid from the pressurefilter that is not recycled is concentrated in a multiple effectevaporator using waste heat from the distillation. The concentratedsyrup from the evaporator is mixed with the solids being sent to thecombustor, and the evaporated condensate is used as relatively cleanrecycle water to the process.

Because the amount of stillage water that can be recycled is limited, anevaporator is included in the process. The total amount of the waterfrom the pressure filter that is directly recycled is set at 25%.Organic salts like ammonium acetate or lactate, steep liquor componentsnot utilized by the organism, or inorganic compounds in the biomass endup in this stream. Recycling too much of this material can result inlevels of ionic strength and osmotic pressures that can be detrimentalto the fermenting organism's efficiency. For the water that is notrecycled, the evaporator concentrates the dissolved solids into a syrupthat can be sent to the combustor, minimizing the load to wastewatertreatment.

Wastewater Treatment

The wastewater treatment section treats process water for reuse toreduce plant makeup water requirements. Wastewater is initially screenedto remove large particles, which are collected in a hopper and sent to alandfill. Screening is followed by anaerobic digestion and aerobicdigestion to digest organic matter in the stream. Anaerobic digestionproduces a biogas stream that is rich in methane that is fed to thecombustor. Aerobic digestion produces a relatively clean water streamfor reuse in the process as well as a sludge that is primarily composedof cell mass. The sludge is also burned in the combustor. This screeningI anaerobic digestion I aerobic digestion scheme is standard within thecurrent ethanol industry and facilities in the 1-5 million gallons perday range can be obtained as “off-the-shelf’ units from vendors.

Combustor, Boiler and Turbo-Generator

The purpose of the combustor, boiler, and turbo-generator subsystem isto burn various by-product streams for steam and electricity generation.For example, some lignin, cellulose, and hemicellulose remainsunconverted through the pretreatment and primary processes. The majorityof wastewater from the process is concentrated to a syrup high insoluble solids. Anaerobic digestion of the remaining wastewater producesa biogas high in methane. Aerobic digestion produces a small amount ofwaste biomass (sludge). Burning these by-product streams to generatesteam and electricity allows the plant to be self-sufficient in energy,reduces solid waste disposal costs, and generates additional revenuethrough sales of excess electricity.

Three primary fuel streams (post-distillate solids, biogas, andevaporator syrup) are fed to a circulating fluidized bed combustor. Thesmall amount of waste biomass (sludge) from wastewater treatment is alsosent to the combustor. A fan moves air into the combustion chamber.Treated water enters the heat exchanger circuit in the combustor and isevaporated and superheated to 510° C. (950° F.) and 86 atm (1265 psia)steam. Flue gas from the combustor preheats the entering combustion airthen enters a baghouse to remove particulates, which are landfilled. Thegas is exhausted through a stack.

A multistage turbine and generator are used to generate electricity.Steam is extracted from the turbine at three different conditions forinjection into the pretreatment reactor and heat exchange indistillation and evaporation. The remaining steam is condensed withcooling water and returned to the boiler feedwater system along withcondensate from the various heat exchangers in the process. Treated wellwater is used as makeup to replace steam used in direct injection.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

In some embodiments, relatively low doses of radiation, optionally,combined with acoustic energy, e.g., ultrasound, are utilized tocrosslink, graft, or otherwise increase the molecular weight of anatural or synthetic carbohydrate-containing material, such as any ofthose materials in any form (e.g., fibrous form) described herein, e.g.,sheared or un-sheared cellulosic or lignocellulosic materials, such ascellulose. The cross-linking, grafting, or otherwise increasing themolecular weight of the natural or synthetic carbohydrate-containingmaterial can be performed in a controlled and predetermined manner byselecting the type or types of radiation employed (e.g., e-beam andultraviolet ore-beam and gamma) and/or dose or number of doses ofradiation applied. Such a material having increased molecular weight canbe useful in making a composite, such as a fiber-resin composite, havingimproved mechanical properties, such as abrasion resistance, compressionstrength, fracture resistance, impact strength, bending strength,tensile modulus, flexural modulus and elongation at break.Cross-linking, grafting, or otherwise increasing the molecular weight ofa selected material can improve the thermal stability of the materialrelative to an un-treated material. Increasing the thermal stability ofthe selected material can allow it to be processed at highertemperatures without degradation. In addition, treating materials withradiation can sterilize the materials, which can reduce their tendencyto rot, e.g., while in a composite. The cross-linking, grafting, orotherwise increasing the molecular weight of a natural or syntheticcarbohydrate-containing material can be performed in a controlled andpredetermined manner for a particular application to provide optimalproperties, such as strength, by selecting the type or types ofradiation employed and/or dose or doses of radiation applied.

When used, the combination of radiation, e.g., low dose radiation, andacoustic energy, e.g., sonic or ultrasonic energy, can improve materialthroughput and/or minimize energy usage.

The resin can be any thermoplastic, thermoset, elastomer, adhesive, ormixtures of these resins. Suitable resins include any resin, or mixtureof resins described herein.

In addition to the resin alone, the material having the increasedmolecular weight can be combined, blended, or added to other materials,such as metals, metal alloys, ceramics (e.g., cement), lignin, organicor inorganic additives, elastomers, asphalts, glass, or mixtures of anyof these and/or resins. When added to cement, fiber-reinforced cementscan be produced having improved mechanical properties, such as theproperties described herein, e.g., compression strength and/or fractureresistance.

Cross-linking, grafting, or otherwise increasing the molecular weight ofa natural or synthetic carbohydrate-containing material utilizingradiation can provide useful materials in many forms and for manyapplications. For example, the carbohydrate-containing material can bein the form of a paper product, such as paper, paper pulp, or papereffluent, particle board, glued lumber laminates, e.g., veneer, orplywood, lumber, e.g., pine, poplar, oak, or even balsa wood lumber.Treating paper, particle board, laminates or lumber, can increase theirmechanical properties, such as their strength. For example, treatingpine lumber with radiation can make a high strength structural material.

When paper is made using radiation, radiation can be utilized at anypoint in its manufacture. For example, the pulp can be irradiated, apressed fiber preform can be irradiated, or the finished paper itselfcan be irradiated. In some embodiments, radiation is applied at morethan one point during the manufacturing process.

For example, a fibrous material that includes a first cellulosic and/orlignocellulosic material having a first molecular weight can beirradiated in a manner to provide a second cellulosic and/orlignocellulosic material having a second molecular weight higher thanthe first molecular weight. For example, if gamma radiation is utilizedas the radiation source, a dose of from about 0.2 Mrad to about 10 Mrad,e.g., from about 0.5 Mrad to about 7.5 Mrad, or from about 2.0 Mrad toabout 5.0 Mrad, can be applied. If e-beam radiation is utilized, asmaller dose can be utilized (relative to gamma radiation), such as adose of from about 0.1 Mrad to about 5 Mrad, e.g., between about 0.2Mrad to about 3 Mrad, or between about 0.25 Mrad and about 2.5 Mrad.After the relatively low dose of radiation, the second cellulosic and/orlignocellulosic material can be combined with a material, such as aresin, and formed into a composite, e.g., by compression molding,injection molding or extrusion. Forming resin-fiber composites isdescribed in WO 2006/102543. Once composites are formed, they can beirradiated to further increase the molecular weight of thecarbohydrate-containing material while in the composite.

Alternatively, a fibrous material that includes a first cellulosicand/or lignocellulosic material having a first molecular weight can becombined with a material, such as a resin, to provide a composite, andthen the composite can be irradiated with a relatively low dose ofradiation so as to provide a second cellulosic and/or lignocellulosicmaterial having a second molecular weight higher than the firstmolecular weight. For example, if gamma radiation is utilized as theradiation source, a dose of from about 1 Mrad to about 10 Mrad can beapplied. Using this approach increases the molecular weight of thematerial while it is with a matrix, such as a resin matrix. In someembodiments, the resin is a cross-linkable resin, and, as such, itcrosslinks as the carbohydrate-containing material increases inmolecular weight, which can provide a synergistic effect to providemaximum mechanical properties to a composite. For example, suchcomposites can have excellent low temperature performance, e.g., havinga reduced tendency to break and/or crack at low temperatures, e.g.,temperatures below 0° C., e.g., below −10° C., −20° C., −40° C., −50°C., −60° C. or even below −100° C., and/or excellent performance at hightemperatures, e.g., capable of maintaining their advantageous mechanicalproperties at relatively high temperature, e.g., at temperatures above100° C., e.g., above 125° C., 150° C., 200° C., 250° C., 300° C., 400°C., or even above 500° C. In addition, such composites can haveexcellent chemical resistance, e.g., resistance to swelling in asolvent, e.g., a hydrocarbon solvent, resistance to chemical attack,e.g., by strong acids, strong bases, strong oxidants (e.g., chlorine orbleach) or reducing agents (e.g., active metals such as sodium andpotassium).

In some embodiments, the resin, or other matrix material, does notcrosslink during irradiation. In some embodiments, additional radiationis applied while the carbohydrate-containing material is within thematrix to further increase the molecular weight of thecarbohydrate-containing material. In some embodiments, the radiationcauses bonds to form between the matrix and the carbohydrate-containingmaterial.

In some embodiments, the carbohydrate-containing material is in the formof fibers. In such embodiments, when the fibers are utilized in acomposite, the fibers can be randomly oriented within the matrix. Inother embodiments, the fibers can be substantially oriented, such as inone, two, three or four directions. If desired, the fibers can becontinuous or discrete.

Any of the following additives can be added to the fibrous materials,densified fibrous materials, or any other materials and compositesdescribed herein. Additives, e.g., in the form of a solid, a liquid or agas, can be added, e.g., to the combination of a fibrous material andresin. Additives include fillers such as calcium carbonate, graphite,wollastonite, mica, glass, fiber glass, silica, and talc; inorganicflame retardants such as alumina trihydrate or magnesium hydroxide;organic flame retardants such as chlorinated or brominated organiccompounds; ground construction waste; ground tire rubber; carbon fibers;or metal fibers or powders (e.g., aluminum, stainless steel). Theseadditives can reinforce, extend, or change electrical, mechanical orcompatibility properties. Other additives include lignin, fragrances,coupling agents, compatibilizers, e.g., maleated polypropylene,processing aids, lubricants, e.g., fluorinated polyethylene,plasticizers, antioxidants, opacifiers, heat stabilizers, colorants,foaming agents, impact modifiers, polymers, e.g., degradable polymers,photostabilizers, biocides, antistatic agents, e.g., stearates orethoxylated fatty acid amines. Suitable antistatic compounds includeconductive carbon blacks, carbon fibers, metal fillers, cationiccompounds, e.g., quaternary ammonium compounds, e.g.,N-(3-chloro-2-hydroxypropyl)-trimethylammonium chloride, alkanolamides,and amines. Representative degradable polymers include polyhydroxyacids, e.g., polylactides, polyglycolides and copolymers of lactic acidand glycolic acid, poly(hydroxybutyric acid), poly(hydroxyvaleric acid),poly[lactide-co-(e-caprolactone)], poly[glycolide-co-(e-caprolactone)],polycarbonates, poly(amino acids), poly(hydroxyalkanoate)s,polyanhydrides, polyorthoesters and blends of these polymers.

When described additives are included, they can be present in amounts,calculated on a dry weight basis, of from below 1 percent to as high as80 percent, based on total weight of the fibrous material. Moretypically, amounts range from between about 0.5 percent to about 50percent by weight, e.g., 5 percent, 10 percent, 20 percent, 30, percentor more, e.g., 40 percent.

Any additives described herein can be encapsulated, e.g., spray dried ormicroencapsulated, e.g., to protect the additives from heat or moistureduring handling.

The fibrous materials, densified fibrous materials, resins or additivesmay be dyed. For example, the fibrous material can be dyed beforecombining with the resin and compounding to form composites. In someembodiments, this dyeing can be helpful in masking or hiding the fibrousmaterial, especially large agglomerations of the fibrous material, inmolded or extruded parts, when this is desired. Such largeagglomerations, when present in relatively high concentrations, can showup as speckles in the surfaces of the molded or extruded parts.

For example, the desired fibrous material can be dyed using an acid dye,direct dye or a reactive dye. Such dyes are available from Spectra Dyes,Kearny, N.J. or Keystone Aniline Corporation, Chicago, Ill. Specificexamples of dyes include SPECTRA™ LIGHT YELLOW 2G, SPECTRACID™ YELLOW4GL CONC 200, SPECTRANYL™ RHODAMINE 8, SPECTRANYL™ NEUTRAL RED B,SPECTRAMINE™ BENZOPERPURINE, SPECTRADIAZO™ BLACK OB, SPECTRAMINE™TURQUOISE G, and SPECTRAMINE™ GREY LVL 200%, each being available fromSpectra Dyes.

In some embodiments, resin color concentrates containing pigments areblended with dyes. When such blends are then compounded with the desiredfibrous material, the fibrous material may be dyed in-situ during thecompounding. Color concentrates are available from Clariant.

It can be advantageous to add a scent or fragrance to the fibrousmaterials, densified fibrous or composites. For example, it can beadvantageous for the composites smell and/or look like natural wood,e.g., cedar wood. For example, the fragrance, e.g., natural woodfragrance, can be compounded into the resin used to make the composite.In some implementations, the fragrance is compounded directly into theresin as an oil. For example, the oil can be compounded into the resinusing a roll mill, e.g., a Banbury® mixer or an extruder, e.g., atwin-screw extruder with counter-rotating screws. An example of aBanbury® mixer is the F-Series Banbury® mixer, manufactured by Farrel.An example of a twin-screw extruder is the WP ZSK 50 MEGAcompunder™,manufactured by Krupp Werner & Pfleiderer. After compounding, thescented resin can be added to the fibrous material and extruded ormolded. Alternatively, master batches of fragrance-filled resins areavailable commercially from International Flavors and Fragrances, underthe trade name Polyiff™ or from the RTP Company. In some embodiments,the amount of fragrance in the composite is between about 0.005% byweight and about 10% by weight, e.g., between about 0.1% and about 5% or0.25% and about 2.5%.

Other natural wood fragrances include evergreen or redwood. Otherfragrances include peppermint, cherry, strawberry, peach, lime,spearmint, cinnamon, anise, basil, bergamot, black pepper, camphor,chamomile, citronella, eucalyptus, pine, fir, geranium, ginger,grapefruit, jasmine, juniperberry, lavender, lemon, mandarin, marjoram,musk, myrhh, orange, patchouli, rose, rosemary, sage, sandalwood, teatree, thyme, wintergreen, ylang ylang, vanilla, new car or mixtures ofthese fragrances. In some embodiments, the amount of fragrance in thefibrous material-fragrance combination is between about 0.005% by weightand about 20% by weight, e.g., between about 0.1% and about 5% or 0.25%and about 2.5%.

While fibrous materials have been described, such as cellulosic andlignocellulosic fibrous materials, other fillers may be used for makingthe composites. For example, inorganic fillers such as calcium carbonate(e.g., precipitated calcium carbonate or natural calcium carbonate),aragonite clay, orthorhombic clays, calcite clay, rhombohedral clays,kaolin, clay, bentonite clay, dicalcium phosphate, tricalcium phosphate,calcium pyrophosphate, insoluble sodium metaphosphate, precipitatedcalcium carbonate, magnesium orthophosphate, trimagnesium phosphate,hydroxyapatites, synthetic apatites, alumina, silica xerogel, metalaluminosilicate complexes, sodium aluminum silicates, zirconiumsilicate, silicon dioxide or combinations of the inorganic additives maybe used. The fillers can have, e.g., a particle size of greater than 1micron, e.g., greater than 2 micron, 5 micron, 10 micron, 25 micron oreven greater than 35 microns.

Nanometer scale fillers can also be used alone, or in combination withfibrous materials of any size and/or shape. The fillers can be in theform of, e.g., a particle, a plate or a fiber. For example, nanometersized clays, silicon and carbon nanotubes, and silicon and carbonnanowires can be used. The filler can have a transverse dimension lessthan 1000 nm, e.g., less than 900 nm, 800 nm, 750 nm, 600 nm, 500 nm,350 nm, 300 nm, 250 nm, 200 nm, less than 100 nm, or even less than 50nm.

In some embodiments, the nano-clay is a montmorillonite. Such clays areavailable from Nanocor, Inc. and Southern Clay products, and have beendescribed in U.S. Pat. Nos. 6,849,680 and 6,737,464. The clays can besurface treated before mixing into, e.g., a resin or a fibrous material.For example, the clay can be surface is treated so that its surface isionic in nature, e.g., cationic or anionic.

Aggregated or agglomerated nanometer scale fillers, or nanometer scalefillers that are assembled into supramolecular structures, e.g.,self-assembled supramolecular structures can also be used. Theaggregated or supramolecular fillers can be open or closed in structure,and can have a variety of shapes, e.g., cage, tube or spherical.

Accordingly, other embodiments are within the scope of the followingclaims.

The invention claimed is:
 1. A mixture comprising: (a) a low molecularweight sugar, (b) a treated cellulosic or lignocellulosic materialhaving a porosity of at least 35% and comprising a plurality ofsaccharide units arranged in a molecular chain, wherein from about 1 outof every 2 to about 1 out of every 250 saccharide units includes acarboxylic acid functional group, (c) water, and (d) a fermentingmicroorganism, wherein the treated cellulosic or lignocellulosicmaterial comprises a cellulosic or lignocellulosic material that hasbeen irradiated with at least 5 Mrad of electron beam radiation, and thecarboxylic acid functional groups act as bonding sites for themicroorganism.
 2. The mixture of claim 1 wherein the material has beenirradiated to provide a first level of radicals and subsequentlyquenched in an oxidizing medium to an extent that the radicals are at asecond level lower than the first level.
 3. The mixture of claim 2wherein the carboxylic acid functional groups are generated by oxidizingthe cellulosic or lignocellulosic material.
 4. The mixture of claim 1wherein the microorganism is a yeast.
 5. The mixture of claim 4 whereinthe yeast is selected from the group consisting of Saccharomycescerevisiae and Pichia stipitis.
 6. The mixture of claim 1 wherein themicroorganism is a bacteria selected from the group consisting ofZymomonas mobilis and Clostridium thermocellum.
 7. The mixture of claim1 wherein the low molecular weight sugar is glucose or xylose.
 8. Themixture of claim 1 wherein the treated cellulosic or lignocellulosicmaterial has a bulk density of less than about 0.5 g/cm3.
 9. The mixtureof claim 1, wherein the cellulosic or lignocellulosic material isselected from the group consisting of paper, paper waste, wood, particleboard, agricultural waste, sewage, silage, grasses, and mixturesthereof.
 10. The mixture of claim 1 further comprising an alcoholselected from the group consisting of methanol, ethanol, propanol,isopropanol, butanol, and mixtures thereof.
 11. The mixture of claim 1wherein the cellulosic or lignocellulosic material has a porosity of atleast 35%.
 12. The mixture of claim 1 wherein the treated cellulosic orlignocellulosic material is in the form of a sheet.
 13. The mixture ofclaim 9 wherein the grasses comprise switchgrass.
 14. The mixture ofclaim 9 wherein the agricultural waste comprises corn stover, corn cobs,or mixtures thereof.
 15. The mixture of claim 9 wherein the agriculturalwaste is selected from the group consisting of rice hulls, bagasse,coconut hair, and mixtures thereof.